JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1992, P. 1042-1044 0095-1137/92/041042-03$02.00/0 Copyright X) 1992, American Society for Microbiology

Vol. 30, No. 4

Evaluation of an Automated Immunodiagnostic Assay System for Direct Detection of Herpes Simplex Virus Antigen in Clinical Specimens SHERYL L. G. JOHNSTON,1t SCOlT HAMILTON,' RANI BINDRA,2 DEBBY A. HURSH,2 AND CURT A. GLEAVES2,3* Bellin Memorial Hospital, Green Bay, Wisconsin 543051'; Fred Hutchinson Cancer Research Institute, Seattle, Washington 981042; and Infectious Disease Laboratory, Chiles Research Institute, Providence Medical Center, Portland, Oregon 972133 Received 21 June 1991/Accepted 23 January 1992

The Vitek ImmunoDiagnostic Assay System (VIDAS) is a 2'/3-h automated qualitative enzyme-linked fluorescent immunoassay developed for the direct detection of herpes simplex virus (HSV) antigen in clinical specimens. A total of 356 clinical specimens submitted for HSV isolation were prospectively evaluated with the VIDAS, and the results of the technique were compared with those of both HSV isolation in cell culture and Herpchek, a nonautomated enzyme immunoassay. Compared to cell culture, VIDAS had a sensitivity of 91.6%Yo and a specificity of 89.3%, with positive and negative predictive values of 82.6 and 95.09%o, respectively. In comparison to Herpchek, VIDAS had a sensitivity of 93.7% and a specificity of 93.09%, with positive and negative predictive values of 89.4 and 95.91%, respectively. The results demonstrated that the VIDAS required minimal manipulation in order to produce results comparable to those of Herpchek and HSV isolation in cell culture.

Over the past decade, commercial industries have introduced various products into the clinical diagnostic virology market in an attempt to make the detection and diagnosis of herpes simplex virus (HSV) infection more rapid. Numerous systems and methods have incorporated the use of monoclonal and polyclonal antibodies, cell culture, and enzyme immunoassay (EIA) systems to identify HSV antigens directly from clinical specimens (1, 3-12, 14, 15). HSV isolation in cell culture is considered the most sensitive method and is the "gold standard" with which all other assay systems are compared for HSV detection (1, 2, 7, 9, 14). VITEK Systems has introduced another EIA-based system for rapid HSV diagnosis with the fully automated Vitek ImmunoDiagnostic Assay System (VIDAS; Vitek Systems Inc., Hazelwood, Mo.). Evaluations were performed at two different sites using the VIDAS for both direct detection of HSV antigen in clinical specimens and culture confirmation of HSV in cell culture. Both standard cell culture and Dupont's Herpchek EIA system were used as the standard of comparison for direct specimen testing. Positive cultures were additionally confirmed and typed by immunofluorescent staining with type-specific monoclonal antibodies. Patient population. Two clinical trial sites were included in this study to provide a diverse patient population. Clinical specimens were submitted to the diagnostic virology laboratories of the Fred Hutchinson Cancer Research Center (FHCR,C), Seattle, Wash. (n = 205), or Bellin Hospital (BH), Green Bay, Wis. (n = 209). FHCRC services a population of patients undergoing bone marrow transplantation, while BH services Planned Parenthood and other medical clinics throughout Wisconsin. A total of 414 clinical specimens were initially collected and prospectively tested for HSV. The criteria for the *

clinical evaluation considered only those specimens collected from vesicular, ulcerated, or crusted lesions to be clinically symptomatic. Therefore, 58 specimens were segregated from the study group because they failed to meet the above criteria. Specimens were collected from oral, genital, and dermal sites. Specimens arrived in the laboratory within 20 h of collection in 2.5 to 3 ml of viral transport medium consisting of modified Eagle medium, antibiotics, and gelatin. Specimens were held at 2 to 4°C if direct transport to the laboratory was delayed more than 30 min. Two cell culture tubes were each inoculated with 0.25 ml of vortexed specimen. Specimens (0.80 ml) were introduced into the VIDAS for direct HSV assay and in duplicate into the DuPont (Dupont Co., North Billerica, Mass.) Herpchek HSV EIA system (0.100 ml per well). VIDAS. The VIDAS HSV assay is an enzyme-linked fluorescent immunoassay and was performed according to the package insert protocol. Briefly, the instrument uses a disposable tip called a solid-phase receptacle (SPR) which is precoated by the manufacturer with rabbit anti-HSV-1 and anti-HSV-2 polyclonal antibodies. The SPR serves as the solid phase of the assay as well as a pipetting device. All of the reagents needed for the assay are contained in a dual sealed reagent strip. The HSV dual reagent strip consists of a sample reference strip and a sample test strip. These strips are identical except that in the sample reference strip biotinylated normal mouse immunoglobulin G is used instead of biotinylated mouse anti-HSV. After the specimen is added manually to the reagent strip (0.4 ml of sample into each side of the dual strip), the completion of the assay is performed automatically by the VIDAS module (13). The sample is automatically cycled in and out of the SPR, where the HSV antigens bind. Following a series of reagent and wash cycles, biotinylated murine monoclonal anti-HSV antibody is drawn into the SPR, where it is cycled in and out of the SPR and will attach to any HSV antigens bound to the SPR wall. More washing steps remove

Corresponding author.

t Present address: Virology Laboratory, St. Vincent Hospital,

P.O. Box 13508, Green Bay, WI 54307-3508.

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unbound antibodies, and streptavidin conjugated to alkaline phosphatase enzyme is then cycled in and out of the SPR. The module then washes the SPR again, and a fluorescent substrate, 4-methylumbelliferyl phosphate, is introduced into the SPR and produces a measurable fluorescence. Nonspecific background fluorescence information is gained from the sample reference side of the dual strip containing the patient sample. The intensity of fluorescence is measured by the optical scanner in the VIDAS. When the VIDAS HSV assay is completed, the results are analyzed automatically by the computer. A test value is calculated from the fluorescence readings of the sample test and sample reference strips. The test value is compared to a set of thresholds, the final result of the assay is interpreted, and a report is printed for each sample. Herpchek. Specimens were also tested with the DuPont Herpchek EIA system according to the manufacturer's protocol, which included the use of Herptran concentrate to complement the use of non-DuPont transport medium (5, 14). The 4-h assay was completed by adding, in duplicate, vortexed specimen to microwells coated with purified rabbit anti-HSV serum. Following the initial 2-h incubation and wash, biotinylated, monoclonal, HSV-specific detector antibody was added, and the plate was incubated for another 30 min. The plate was next washed, and streptavidin-horseradish peroxidase was added. The plate was then incubated 15 min, and the o-phenylenediamine substrate was added. Following another 1-h incubation, the optical density was read by a spectrophotometer (490 and 620 nm), and a cutoff value was calculated by adding 0.07 to the mean of the optical density values of the negative viral transport medium controls (3, 5). Cell culture. Locally produced rhabdomyosarcoma and mink lung cells were inoculated with specimens at BH, and A549 and HF cells were inoculated at FHCRC. The cell cultures were observed daily for 7 to 10 days for cytopathic effect. Positive cultures were scraped and stained for confirmation and typing by using type-specific monoclonal antibodies (Syva Co., Palo Alta, Calif.). A total of 356 patient specimens fit the clinical trial protocol and were directly tested on the VIDAS and Herpchek systems and inoculated into cell culture. There were 125 positive cultures (45 stained for type 1, 79 stained for type 2, and 1 sample stained for both types 1 and 2). The VIDAS detected 109 (87.2%) of the 125 positive cultures. Of the remaining 16 positive cultures, 3 of these were considered VIDAS invalid, 3 were considered equivocal, and 10 were considered negative by VIDAS. Of the 231 negative cultures, the VIDAS identified 192 as negative, 23 direct specimens as positive, and 16 as equivocal. Herpchek identified 114 (91.2%) of the 125 positive cultures. Of the 11 remaining samples, 8 were identified as negative and 3 were identified as equivocal by Herpchek. In the 231 negative cultures, 205 were identified as negative, 19 were considered positive, and 7 were considered equivocal by Herpchek. Upon removal of VIDAS equivocal and invalid results, the VIDAS in comparison to cell culture as the standard (n = 334, which excludes 19 equivocal and 3 invalid results) had a sensitivity of 91.6% and a specificity of 89.3%, with a positive predictive value (PPV) of 82.6% and a negative predictive value (NPV) of 95.0%. When the same direct VIDAS data was compared with data from Herpchek EIA as the standard, the sensitivity was 93.7%, the specificity was 93.0%, the PPV was 89.4%, and the NPV was 95.9%.

NOTES

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TABLE 1. Intertest comparison of cell culture, VIDAS, and Herpchek EIA for the detection of HSV Test and standard

VIDAS direct Herpchek Culture Culture and/or

%a No. of samples Positive or negative Tested Sens Spec PPV NPV by VIDAS

Agr

325

346

93.7 93.0 89.4 95.9 93.2

334 334

356 356

91.6 89.3 82.6 95.0 90.1 92.3 94.1 90.9 95.0 93.4

325

334

89.4 95.9 93.7 93.0 93.2

346

356

93.4 91.5 85.7 96.2 92.2

Herpchek Herpchek direct VIDAS Culture

a Sens, sensitivity; Spec, specificity; PPV, positive predictive value; NPV, negative predictive value; Agr, agreement.

The Herpchek EIA compared with culture as the standard (n = 346, which excludes 10 Herpchek equivocal results) had a sensitivity of 93.4%, a specificity of 91.5%, a PPV of 85.7%, and a NPV of 96.2% (Table 1). To date, numerous commercial companies have placed an array of EIA kits on the market which detect HSV antigen directly from patient specimens. While these assays are "technique" sensitive, their limitation is a lack of automation. The VIDAS is, however, completely automated. Like other commercially available nonculture tests for HSV, the VIDAS assay requires specimens from symptomatic lesions which are necessary for the most sensitive and specific results (2). Comparisons were made between the VIDAS, an automated system; Herpchek, a manual EIA system; and standard cell culture. Initial data were collected on a wide array of lesions, including rashes and macular and papular sites. Since reports indicate that rapid assays are most sensitive when samples are taken from highly symptomatic, mucocutaneous lesions, the final analysis of the clinical trial data was made by using only vesicular, ulcerated, or crusted lesions (2). The initial 414 direct specimens did yield a lower sensitivity (81.3%), a lower specificity (81.2%), and a lower PPV (80%) and NPV (92%) than the data showed after the nonspecific lesion types and unresolved samples were removed. Using only symptomatic lesions (as the protocol required), the VIDAS performed nearly as well as the Herpchek system. However, there were twice as many samples unresolved by using the VIDAS as compared to the Herpchek assay. This includes three samples that were culture and Herpchek positive but were considered invalid by VIDAS. If one includes all 414 samples originally tested, the Herpchek EIA detected 127 of 139 (91.4%) total culture-positive samples, where as the VIDAS detected 113 of 139 (81.3%) total culture-positive samples. Currently, both systems are approved by the Food and Drug Administration and recommended for use on only symptomatic specimens. While it is ideal to obtain only these highly symptomatic lesion types for rapid assays, many times specimens which are undefined as to lesion type or from asymptomatic patients arrive in the laboratory. The data herein would suggest that the VIDAS test would not be beneficial with these types of samples. However, there are data to support that sensitivity is minimally altered, as

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NOTES

compared with that of cell culture, when asymptomatic specimens are used in the Herpchek assay (5). Cell culture backup is recommended for negative HSV results screened with the VIDAS, but this recommendation is not made for the Herpchek assay. We agree with the recent statements made by Gonik and colleagues (6) that cell culture may not be 100% sensitive for HSV detection. Gonik further states that technical errors and viral inoculum size can significantly affect cell culture. This is correct, but these same conditions can significantly affect EIAs as well (2, 3, 5, 6). As is evident in this study as well as others, for optimal HSV detection both cell culture and EIA may need to be performed (3, 5, 9, 12, 15). The Herpchek system was able to run a higher volume of specimens at one time (47 in duplicate with 2 controls) than the VIDAS (13 with 2 controls) but required over 4 h to run and excessive hands-on time for setup and processing in comparison to the VIDAS system, which took less than 3 h to run in completion. Both systems are designed to run fewer specimens if necessary. The Herpchek system has breakaway strips of 8 wells which allow for fewer than 47 tests to be run at a time. The VIDAS independent strips could run fewer specimens or simultaneously run combinations of specimens for viruses other than HSV (13). The VIDAS system is fully automated not only in the internal wash and incubation steps, but also in the final result calculations. Herpchek requires manually run washes, reagent additions, and mathematical result calculations. When culture confirmation was performed (data not shown) with the VIDAS, it was not highly specific when compared to confirmation by cell culture scrapedown and staining with specific monoclonal antibodies. Furthermore, the cost and time involved in using the VIDAS for culture confirmation were not warranted. It took nearly 3 h to complete a culture confirmation using the VIDAS at a cost of several dollars. To confirm HSV cytopathic effect, which is highly recognizable, with monoclonal antibody stain the cost of material was less than 1 dollar, with a completion time of only an hour. Typing of the positive cultures was also available with the use of monoclonal antibody stains. Neither the VIDAS nor Herpchek was able to provide typing information from the direct specimen analysis or upon culture confirmation. HSV typing is not, however, a requirement for HSV diagnosis. On the basis of these evaluations, the VIDAS HSV assay has a sensitivity and specificity similar to those of Herpchek and is more rapid than either the Herpchek assay or cell culture for the diagnosis of HSV infection from symptomatic samples. However, the VIDAS is limited to just 13 samples per run compared with a 96-well format for the Herpchek assay. Each EIA assay was restricted because of equivocal results which need to be resolved before being reported to the physician. This problem was more apparent with the VIDAS than with the Herpchek assay. Both EIA systems, however, can detect nonviable HSV antigen which cannot be confirmed with culture, and this could be beneficial in monitoring antiviral therapy as well as specimens that have delayed transport times (6). Currently, HSV can be identified in conventional cell culture rapidly, with equal, if not greater, sensitivity and

specificity and at a lower cost than with either of the two EIA systems tested. Each laboratory must consider its needs on the basis of patient population, pricing, and technical help to determine what is best for its specific environment. REFERENCES 1. Baker, D. A., B. Gonik, P. 0. Milch, A. Berkowitz, S. Lipson, and U. 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. Corey, L. 1986. Laboratory diagnosis of herpes simplex virus infections, principles guiding the development of rapid diagnostic tests. Diagn. Microbiol. Infect. Dis. 4:111S-119S. 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. 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. 5. Gleaves, C. A., D. H. Rice, and C. F. Lee. 1990. Evaluation of an enzyme immunoassay for the detection of herpes simplex virus (HSV) antigen from clinical specimens in viral transport media. J. Virol. Methods 28:133-140. 6. Gonik, B., M. Seibel, A. Berkowitz, M. B. Woodin, and K. Mills. 1991. Comparison of two enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen. J. Clin. Microbiol. 29:436-438. 7. Halstead, D. C., D. G. Beckwith, R. L. Sautter, L. Plosila, and K. A. Schneck. 1987. Evaluation of a rapid latex slide agglutination test for herpes simplex virus as a specimen screen and culture identification method. J. Clin. Microbiol. 25:936-937. 8. Hussain Qadri, S. M., S. G. M. Qadri, Y. Khan, T. J. McGarry, and M. N. Al-Ahdal. 1988. Detection of herpes simplex virus by biotinylated DNA probes. Diagn. Microbiol. Infect. Dis. 11: 145-149. 9. 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. 10. Lafferty, W. E., S. Krofft, M. Remington, R. Giddings, C. Winter, A. Cent, and L. Corey. 1987. Diagnosis of herpes simplex virus by direct immunofluorescence and viral isolation from samples of external genital lesions in a high-prevalence population. J. Clin. Microbiol. 25:323-326. 11. MacDonald, R. L., B. L. Hughes, S. L. Aarnaes, E. M. Peterson, and L. M. de la Maza. 1988. Evaluation of a shell vial centrifugation method for the detection of herpes simplex virus. Diagn. Microbiol. Infect. Dis. 9:51-54. 12. Patel, H., L. D. Frenkel, M. Greenhalgh, R. Howell, and S. Patel. 1991. Rapid culture confirmation of herpes simplex virus by a monoclonal antibody-based enzyme immunoassay. J. Clin. Microbiol. 29:410-412. 13. Rogers, C. H., K. L. Hoffman, and R. M. Juris. 1989. An automated system for infectious disease diagnostics. Am. Clin. Lab. 8:35-37. 14. Verano, L., and F. J. Michalski. 1990. Herpes simplex virus antigen direct detection in standard virus transport medium by Du Pont Herpchek enzyme-linked immunosorbent assay. J. Clin. Microbiol. 28:2555-2558. 15. Warford, A. L., R. A. Levey, C. A. Strong, and K. A. Rekrut. 1986. Comparison of two commercial enzyme-linked immunosorbent assays for detection of herpes simplex virus antigen. Am. J. Clin. Pathol. 85:229-232.

Evaluation of an automated immunodiagnostic assay system for direct detection of herpes simplex virus antigen in clinical specimens.

The Vitek ImmunoDiagnostic Assay System (VIDAS) is a 2 1/3-h automated qualitative enzyme-linked fluorescent immunoassay developed for the direct dete...
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