ELVIS: Title and subTitle BreakA New 24-Hour Culture Test for Detecting Herpes Simplex Virus From Ocular Samples

HERPES SIMPLEX virus (HSV) infection of the cornea is a potentially blinding disease that must be diagnosed and treated promptly. Classical HSV keratitis presents as epithelial disease in the form of a corneal dendrite or geographical ulcer on slitlamp examination. We previously reported that initial clinical diagnosis is only 55% to 65% accurate based on a positive cell culture.^{1 - 2} It is essential that patients be diagnosed and treated with antiviral drugs at the earliest onset of HSV infection to eliminate replicating virus. It is also important to rule out live virus in those patients with herpetic stromal keratitis who are being treated with anti-inflammatory drugs (eg, prednisolone acetate).

In our laboratory, ocular specimens are tested routinely for replicating HSV by cell culture and for the presence of HSV antigens using a 4-hour enzyme immunoassay, Herpchek (NEN, Boston, Mass).² Diagnosis based on a positive cell culture can delay crucial therapy in atypical disease because the detection of cytopathic effects generally requires more than 2 days. Herpchek testing can be processed within 24 hours, but single-patient testing may not be cost effective since many laboratories can not dedicate 4 hours of technical time for one test. ELVIS (Enzyme Linked Virus Inducible System) (BioWhittaker, Walkersville, Md) is a new simple HSV detection test that can detect live HSV in a specially engineered cell line within 24 hours and is more economical for single-patient testing since only 1 hour of technical time is required.^{3 - 4} In this study, we evaluated ELVIS to detect HSV ocular disease, in comparison with standard cell culture and Herpchek.

ELVIS was compared with standard cell culture and Herpchek in retrospective and prospective studies using patient ocular specimens. In the retrospective study, ELVIS was processed on clinical samples with known positive cell cultures and Herpchek results. In the prospective study, ocular samples collected from patients with suspected HSV ocular infection were tested for HSV with cell culture, ELVIS, and Herpchek. All testing was processed from a single ocular specimen from each patient.

Patient samples were obtained from the cornea and/or conjunctiva with soft-tipped applicators, spatulas, and/or jeweler's forceps. The specimens were collected in 2.0 mL of ChlamTrans transport medium (Bartels Inc, Issaquah, Wash). Monolayers of A549 (human lung carcinoma) cells were pretreated with phosphate-buffered saline containing calcium and magnesium, and inoculated with 0.5 mL of patient sample. The monolayers were examined every other day for 3 weeks for the presence of viral cytopathic effect. Herpes simplex virus' cytopathic effect was confirmed for HSV antigens using Herpchek.

ELVIS technology generates an endogenous, intracellular accumulation of the bacterial enzyme β-galactosidase in HSV-infected cells. After the cell culture amplification period, the cell monolayer is fixed and incubated in a staining buffer containing substrate for the HSV-induced enzyme that is specific to herpes simplex, both types 1 and 2. After the staining period, the monolayers are examined by light microscopy 1 to 5 hours later to detect positive blue-stained cells (Figure 1).

The retrospective study was biased in that Herpchek-negative, culture-positive samples were specifically selected to test whether ELVIS could detect HSV that Herpchek had missed. This was necessary to validate testing in the clinical laboratory for proficiency and billing reimbursement. ELVIS tests were processed according to the recommended procedure,⁵ concurrently with A549 cell culture isolation for 36 true-positive frozen-stock ocular samples that were cell culture positive for HSV, and 25 true-negative samples(5 varicella zoster virus [75% CYTOPATHIC EFFECT inoculum], 5 Staphylococcus aureus [10⁵ colony-forming units per milliliter], 5 Pseudomonas aeruginosa [10⁵ colony-forming units per milliter], 5 Streptococcus viridans [10⁵ colony-forming units per milliliter], and 5 adenovirus serotype 5[10³ colony-forming units per milliliter]). Cell culture isolation was processed on the previously positive samples to assure that live virus was present in the stored frozen stocks. The true-negative samples were also prepared in 2.0 mL of ChlamTrans transport medium. The frozen-stock samples were collected and stored between September 1992 through July 2000, in the anonymous ophthalmic microbiology tissue bank as approved by the institutional review board at the University of Pittsburgh (Pittsburgh, Pa; IRB 000943).

In brief, shell vials of ELVIS cells were incubated at 35°C overnight prior to inoculation. The maintenance medium was discarded from the cells, and 0.2 mL of specimen was added to each shell vial (one vial per patient). A positive HSV control and a negative control were included. The shell vials were centrifuged at 900g for 60 minutes at room temperature(22°C), and 1.0 mL of replacement medium was added to each shell vial. The shell vials were incubated for 24 hours at 35°C. The replacement medium was discarded from the cells, and 0.25 mL of cell fixative was added to each shell vial. The cell fixative was discarded after 10 minutes, and 0.25 mL of staining substrate buffer was added to each shell vial. The shell vials were incubated at 35°C and examined hourly for 5 hours for blue color development using a light microscope, with original magnification at ×40 to ×100. Any cells demonstrating a blue color development were considered positive for HSV (Figure 1). Cells with no blue color development were deemed negative for HSV.

The retrospective study validated the ELVIS test as a routine test for detecting HSV in ocular specimens. As a laboratory testing battery, all ocular specimens submitted for ocular HSV testing from September 2000 to January 2002 were processed by ELVIS, standard cell culture, and Herpchek. The testing data were tabulated anonymously without regard to patient personal information.

The Herpchek Direct HSV Antigen Test is a forward sandwich enzyme immunoassay that uses purified rabbit polyclonal anti-HSV antibodies for the capture of the HSV antigens. The capture antibody has been immobilized onto the interior surface of the microtiter plate wells. An appropriate volume of the clinical specimen is incubated in the coated plate well for the binding of the HSV antigens onto the solid phase. The immobilized antigen is then made to react with a second reagent (biotinylated mouse monoclonal anti-HSV). The amount of biotinylated antibody bound to the antigen is measured by creating a complex with streptavidin-horseradish peroxidase (HRP) conjugate, which catalyzes the conversion of a chromogenic substrate (o-phenylenediamine) into a colored (yellow) product. The yellow reaction product is detected visually and indicates the presence of HSV antigens in the sample. For the retrospective and prospective studies, all collected ocular samples suspicious for HSV were processed at the time of the initial examination using Herpchek.⁶

The retrospective study determined that the ELVIS test based on positive cell culture was 86.1% sensitive (31/36), 100% specific (25/25), and 91.8% efficient (56/61). The positive predictive value was 100% (31/31), and the negative predictive value was 83.3% (25/30). Herpchek, based on positive cell culture, was 80.5% sensitive (29/36). The sensitivities of ELVIS and Herpchek were statistically equivalent (χ², P= .53). Three ocular specimens were ELVIS positive and Herpchek negative. One ocular sample was ELVIS negative and Herpchek positive.

In the prospective study, 422 patients had ocular samples tested for the presence of HSV. Of these, 8.3% (35/422) tested positive for the presence of HSV. Of the patients positive for HSV, 33 were cell culture positive, and 2 were cell culture negative and ELVIS negative, but Herpchek positive. One cell culture positive sample was ELVIS negative and Herpchek positive. One cell culture positive sample was ELVIS positive and Herpchek negative. The mean ± SD time to finding a positive culture was 2.85 ± 2.21 days (range, 1-8 days). ELVIS and Herpchek, based on positive cell culture, were both 84.8% sensitive (28/33).

The cumulative sensitivities of ELVIS and Herpchek, from the retrospective and prospective studies, were statistically equivalent (χ²₁, P = .64) at 85.5% (59/69) and 82.6% (57/69), respectively.

Diagnostic tests should be timely, so that they provide pertinent information that guides appropriate therapy. Laboratory tests to detect HSV from ocular specimens should be completed within 24 hours, but the economic restraints at some institutions have limited the work force on weekends to essential duty, and many tests are now delayed to the following Monday or batched (waiting to test multiple samples instead of one) for cost effectiveness. Standard cell culture will generally not provide final HSV results within 24 hours, and the Herpchek is too time consuming (4 hours) to be processed on a single patient during weekends and nonworking hours. ELVIS is simple enough to be processed within 1 hour at the end of a working day and completed the next morning. ELVIS is well suited for the small laboratory that processes tests daily on single patients, especially on weekends. Positive tests are easy to detect visually, but patience and time should be allotted to examine the entire monolayer since a single blue-stained cell denotes the presence of HSV. We presently examine all monolayers after an additional 24 hours of incubation for deeper staining of single cells that may not have been as apparent on the first day.

Our retrospective and prospective studies determined that ELVIS is not 100% sensitive, thus standard cell culture should be used to confirm negative tests. The sensitivity of Herpchek in our study was biased because we selected for negative results to test whether ELVIS would provide positive results for these samples. We reported⁷ that the Herpchek was 90.2% sensitive for detecting HSV throughout a 7-year period. As the sensitivity of Herpchek was lower in the current study (84.8%), but equivalent to ELVIS, the true sensitivity of ELVIS may actually be higher than reported in this study. ELVIS should not replace Herpchek because 2 patients in this study were Herpchek positive, ELVIS negative, and cell culture negative. Herpchek only requires the presence of antigens, and not live virus. This may be important in patients who were previously treated with antiviral drugs that inactivated the virus but did not eliminate the antigen.

In conclusion, ELVIS is an easy HSV diagnostic test that can provide positive answers within 24 hours, but it requires confirmation by cell culture when tests are negative. ELVIS is well suited for the small laboratory, and it complements Herpchek and standard cell culture for providing definitive HSV testing.

Submitted for publication January 31, 2002; final revision received March 11, 2002; accepted March 27, 2002.

This study was generously supported by the Pennsylvania Sight Conservation and Eye Research Foundations Inc (Feasterville, Pa), the Eye and Ear Foundation of Pittsburgh (Pittsburgh, Pa), core grant EY 08098 for vision research from Research to Prevent Blindness Inc (New York, NY), and BioWhittaker Inc (Walkersville, Md).

This study was presented in part as a poster presentation at the American Society of Microbiology meeting, Orlando, Fla, May 22, 2001; and at the 2002 ARVO meeting in Ft Lauderdale, Fla.

Corresponding author: Regis P. Kowalski, MS, M(ASCP), Department of Ophthalmic Microbiology, Eye and Ear Institute Bldg, 203 Lothrop St, Pittsburgh, PA 15213 (e-mail: kowalskirp@msx.upmc.edu).