Biotin-avidin-amplified enzyme immunoassay for detection of herpes simplex virus antigen in clinical specimens

A biotin-avidin-amplified enzyme-linked immunosorbent assay (B-A ELISA) has been developed to detect herpes simplex virus type 1 (HSV-1) and HSV-2 antigens in clinical specimens. The test was designed as a solid-phase, double-antibody, sandwich assay in which plates were coated with a polyclonal rabbit immunoglobulin G anti-HSV reagent, and the sandwich antibody was a biotin-labeled mouse immunoglobulin M monoclonal antibody that reacts with a common antigen associated with HSV-1 and HSV-2. The test can be completed in 4 h if antibody-coated plates are available. The detection limit of the B-A ELISA, determined by titration of virus stocks, was found to be approximately 90 PFU or 6 X 10(3) physical particles of either HSV-1 or HSV-2 per 50 microliter of virus stock. The following results were obtained in a study in which swabs were taken from a variety of lesions and assayed for infectivity in tissue culture and by B-A ELISA. Of 421 suspected HSV lesions tested, 69 were positive by both tests and 159 were negative by both tests. A total of 122 were positive by B-A ELISA but negative for infectivity. Seventy-one were negative by B-A ELISA but contained infectious virus. The HSV specificity of the assay was substantiated by partial blocking of reactivity with rabbit immunoglobulin G anti-HSV and by the absence of reactivity with a nonspecific biotin-labeled mouse immunoglobulin M monoclonal antibody.     

Evaluation of a visual, rapid, membrane enzyme immunoassay for the detection of herpes simplex virus antigen.

We evaluated a 12-min, direct, monoclonal antibody-based enzyme immunoassay (EIA) (SureCell; Kodak, Rochester, N.Y.) which aids in the detection of herpes simplex virus infection; the assay system is also approved for culture confirmation. The test was evaluated from direct clinical samples and compared with conventional culture methodology by using a single swab. A total of 265 specimens from 180 female cervical-urogenital sites, 62 male urogenital sites, 4 rectal sites, 3 skin sites, 6 oral sites, and 10 colposcopy sites were collected on Dacron or cotton swabs and placed in viral transport medium (VTM). Within 6 h of receipt, 0.2 ml of the vortexed VTM was inoculated into each of two replicate cell cultures. Cell monolayers were observed daily for ten days, and cytopathic effect was confirmed by using an indirect immunoperoxidase reagent. The procedure for the SureCell assay conformed to the manufacturer's recommendations. When conventional culture was compared with EIA results, the overall sensitivity, specificity, positive predictive value, negative predictive value, and agreement were 64.4, 98.9, 96.7, 84.4, and 87.2%, respectively. Variables affecting the EIA sensitivity are the stage of the lesion and conventional culture methodologies. A review of culture results for 32 EIA false-negative tests indicated that 15 were detected after 48 h of incubation. Cytopathic effect observed at 48-, 72-, and 96-h cutoffs altered the sensitivity for the EIA. To ensure detection of SureCell herpes simplex virus-negative specimens, it is recommended that an unused aliquot of VTM be tested in cell culture.     

Evaluation of an enzyme immunoassay for the detection of herpes simplex virus (HSV) antigen from clinical specimens in viral transport media

A total of 301 clinical specimens (229 culture positive and 72 culture negative) were assayed retrospectively by an enzyme immunoassay. Specimens were transported to the virus lab in viral transport media (VTM), and were inoculated into HF and A549 cell culture tubes for viral isolation with the remainder of the sample being saved at -70 degrees C. Specimens were thawed, vortexed and resuspended in 10 x Herptran concentrate and each sample was then added in duplicate to designated wells of a microtiter plate for the EIA assay. The EIA detected 147/150 (98%) culture positive specimens from symptomatic patients, 63/79 (79.7%) culture positive specimens from patients considered asymptomatic and 210/229 (91.7%) culture positive specimens overall. The EIA was negative for 70/72 (97.2%) culture negative specimens. These data suggest that the EIA test can be used with clinical specimens submitted in conventional VTM. However, VTM samples which are EIA negative, particularly with EIA values close to the EIA positive cutoff value, need to be cultured.     

Comparison of cell culture with an amplified enzyme immunoassay for diagnosing genital herpes simplex infection.

An amplified enzyme immunoassay (EIA) for herpes simplex virus (Novo Nordisk) was compared with cell culture in 853 genital specimens from a genito-urinary medicine clinic. The sensitivity of the EIA was 86% and its specificity 99.6%. The sensitivity increased to 94% for lesion swabs but decreased to 68% for cervical swabs. Sensitivity for urethral and vulval swabs was 83% and 82%, respectively. It is concluded that the EIA is specific and quick and easy to perform. It will be suitable for testing for genital herpes simplex infections in laboratories without access to local cell culture facilities.     

Capillary enzyme immunoassay for rapid detection of herpes simplex virus in clinical specimens.

Capillary enzyme immunosorbent assay (CapELISA) is a modification of the standard enzyme immunosorbent assay which permits rapid detection of viral antigens in clinical specimens. The capillary tube format provides a very large reactive surface relative to the sample size. The close proximity of antigen to antibody in the tube optimizes the reaction, resulting in increased sensitivity and shorter incubation requirements. Sensitivity is further enhanced by use of a biotin-avidin enzyme detector system and a fluorogenic rather than a colorigenic substrate. The assay is performed at ambient temperature and requires less than 2 h. It is read with an inexpensive hand-held black light. Data for the use of CapELISA for the detection of herpes simplex virus in clinical specimens are presented. The results show greater than or equal to 85% sensitivity and 100% specificity when compared with tissue culture tests on the same samples. This new system should be advantageous for the diagnosis, treatment, and management of patients with herpesvirus infections and for the prevention of neonatal herpes acquired by passage of the fetus through an infected birth canal.     

Diagnosis of herpes simplex virus infection in a clinical setting by a direct antigen detection enzyme immunoassay kit.

A commercial 4-h direct herpes simplex virus (HSV) antigen detection enzyme immunoassay (EIA) kit (Du Pont Herpchek) was evaluated by using 273 clinical specimens obtained in a hospital-based infectious disease practice. The EIA was compared with a standard culture method in which WI38 cells were inoculated within 20 min of sample collection. Cultures were observed for 2 weeks, and positive findings were confirmed by fluorescein-labeled monoclonal antibody (FA) staining. The values for the overall HSV detection rate were 40.7% by the standard culture method and 41.4% by EIA. In eight cases, the EIA was positive, while the culture method was negative; however, clinical data and confirmatory blocking EIA suggested that a true HSV infection was present. For six FA-confirmed, culture-positive samples, the direct EIA was negative; however, an EIA performed on the supernatants of these cultures was positive, suggesting that the failure of the EIA to detect these samples was not due to lack of strain specificity of the test. After confirmatory tests of standard culture and EIA discrepant results, the overall sensitivity of the test was 95.0% (113 of 119) and the specificity was 100% (154 of 154).     

Evaluation of an enzyme-linked immunosorbent assay for the detection of herpes simplex virus antigen.

An enzyme-linked immunosorbent assay (ELISA) kit for herpes simplex virus developed by Ortho Diagnostic Systems, Inc., was evaluated. In phase I experiments, 263 clinical specimens from genital lesions were extracted into serum-free medium and then tested by ELISA for herpes simplex virus antigen. The results were compared with those obtained by conventional viral culture. Of 83 specimens, 65 were positive by ELISA (sensitivity, 78.3%). In phase II experiments, 249 clinical specimens were tested for herpes simplex virus antigen in direct specimen and in cell cultures (MRC-5 and rabbit kidney) incubated for 2, 4, and 7 days. Of 63 specimens, 40 were positive by ELISA in the direct specimen (sensitivity, 63.5%), and by 7 days incubation, 100% of the cultures positive by viral cell culture were also positive by ELISA. The ELISA was reproducible, and when both the direct detection and amplification culture were used, the sensitivity of ELISA paralleled the diagnosis of herpes simplex virus infections by viral cytopathic effect.     

Evaluation of Syva enzyme immunoassay for detection of Chlamydia trachomatis in genital specimens.

Detection of Chlamydia trachomatis infection was evaluated by culture and a new Syva enzyme immunoassay (EIA) in 1,012 patients at two Baltimore, Md., sexually transmitted disease clinics. The overall chlamydia prevalence determined by culture was 12%. For 506 fresh cervical and urethral specimens, the sensitivity of Syva EIA was 90% and its specificity was 94% compared with culture. Discordant Syva EIA results were further evaluated by staining the sediment in centrifuged culture transport media and Syva EIA transport tubes with a fluorescent monoclonal antibody to C. trachomatis to detect elementary bodies. Reanalysis of the data after use of this technique to resolve discordant results increased sensitivity and specificity to 92 and 96%, respectively. A subsample of 307 fresh cervical specimens was also tested in a three-way comparison using Abbott Chlamydiazyme, Syva EIA, and culture. In this sample, compared with culture, the sensitivity and specificity of Syva EIA were 87 and 95%, respectively, and for Chlamydiazyme they were 77 and 98%, respectively. Syva EIA is a 4-h, easy-to-perform enzyme-linked immunosorbent assay which has a high sensitivity with fresh genital specimens and offers an excellent alternative to culture.     

Evaluation of biotin-streptavidin enzyme-linked immunosorbent assay for detection of genital herpes simplex virus infection

A biotin-streptavidin enzyme-linked immunosorbent assay (B-SA ELISA) was evaluated for detection of herpes simplex virus (HSV) in clinical specimens which were cervico-vaginal swabs from 205 asymptomatic women and swabs from the genital lesions of 163 suspected patients. All specimens were also subjected to a conventional virus isolation in cell culture. A blocking B-SA ELISA had 100% specificity and 98% sensitivity compared with viral isolation from patients, but had only 40% sensitivity using specimens from asymptomatics. The conventional B-SA ELISA might also be used; it gave results corresponding to B-SA ELISA blocking test except for a single specimen which was considered a false positive.     

Evaluation of an enzyme-linked immunoassay employing a covalently bound capture antibody for direct detection of herpes simplex virus.

The FDL enzyme-linked immunosorbent assay (ELISA; Fairleigh Dickinson Laboratories, Inc., Abilene, Tex.) for the detection of herpes simplex virus (HSV) makes use of a covalently attached antibody. This assay was compared with viral isolation and with the Ortho HSV Antigen Detection ELISA (Ortho Diagnostic Systems, Inc., Raritan, N.J.). One hundred forty-eight specimens were tested. The FDL ELISA identified 66 of 104 specimens from which HSV was isolated, yielding a sensitivity of 63% and a specificity of 95%. These results compared favorably with those obtained by using the Ortho ELISA. The total test time was shorter and the washing step was simpler than that with the Ortho assay, making the FDL assay an attractive alternative to similar methodologies.     

Comparison of a direct antigen enzyme immunoassay, Herpchek, with cell culture for detection of herpes simplex virus from clinical specimens.

Direct enzyme immunoassay (Herpchek) was compared with culture on 21,522 specimens mainly from asymptomatic women for herpes simplex virus detection. Sensitivity and specificity were 73.8 and 97.7%, respectively. The 33% detection rate by enzyme immunoassay in 5 h increased to 43% when the enzyme immunoassay was combined with culture. Herpchek alone was not sensitive enough, but in combination with culture, maximum detection was achieved.     

Evaluation of three methods for typing herpes simpex viras

Three methods of typing herpes simplex virus were compared. A total of 111 clinical isolates obtained from patients not treated with antivirals and seven resistant mutants selected in vitro were tested by immunofluorescence assay using antibodies against herpes simplex virus types 1 and 2. Twenty-nine isolates were also studied by restriction endonuclease analysis. The sensitivity of isolates and resistant mutants to (E)-5-(2-bromovinyl)-2-deoxyuridine was determined. Although a clear difference between the 50 % inhibitory dose for type 1 and type 2 isolates was observed, some drug-resistant mutants might be misidentified by this method.     

Evaluation of an enzyme immunoassay for detection of cryptococcal capsular polysaccharide antigen in serum and cerebrospinal fluid.

The Premier enzyme immunoassay (Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared with a latex agglutination assay (CALAS; Meridian) for the ability to detect cryptococcal capsular polysaccharide antigen (CrAg) in serum and cerebrospinal fluid (CSF). A total of 594 specimens (471 serum samples and 123 CSF samples) obtained from 430 patients, most of whom were at risk for or had AIDS, were tested in parallel by both systems. Both tests were independently evaluated for their ability to (i) detect CrAg when used as a screening test and (ii) quantitate the CrAg present when used as a titration assay. Chart review to assess clinical outcome after the time of specimen collection was conducted for all patients. When both assays were used as screening assays, 103 serum samples and 18 CSF samples were positive and 356 serum samples and 104 CSF specimens were negative by both assays (97.8% concordance). Thirteen specimens (12 serum samples, 1 CSF sample) gave discrepant screening results. When the tests were used as semiquantitative assays for titer determinations, the CrAg titers determined by the enzyme immunoassay were generally higher than those obtained with the latex agglutination assay. In summary, results obtained with the enzyme immunoassay correlated well with those obtained with the latex agglutination test for screening for the presence of CrAg and for determining the titer of CrAg in serum or CSF.     

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 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% and a specificity of 89.3%, with positive and negative predictive values of 82.6 and 95.0%, respectively. In comparison to Herpchek, VIDAS had a sensitivity of 93.7% and a specificity of 93.0%, with positive and negative predictive values of 89.4 and 95.9%, 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.     

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of herpes simplex virus antigen.

A commercial enzyme-linked immunosorbent assay (ELISA) for detection of herpes simplex virus (HSV) antigen in clinical specimens and culture lysates was evaluated. A total of 1,155 specimens and an additional 335 cell culture lysates were tested by ELISA, and results were compared to those obtained by cell culture in primary rabbit kidney, human foreskin, and MRC-5. The sensitivity and specificity of the direct test were 52.5 and 96.9% and those of the culture lysate test were 98.7 and 96.9%, respectively. The sensitivity of the direct ELISA correlated with early cytopathic effect in cell culture and varied with specimen source, ranging from 100% with skin lesions to 40.9% with cervical swabs. Of 60 cervical specimens from asymptomatic individuals, 22 (36.6%) yielded false-positives, which may be due to noninfectious HSV. No reproducible cross-reactions were found with other viruses isolated. The Ortho HSV ELISA was found to be rapid, sensitive, and specific for detection of HSV from cell culture lysates, but it needs reevaluation for direct specimen testing, in particular for screening of asymptomatic obstetrical patients.     

Evaluation of two rapid methods for the detection of herpes simplex virus antigen in patient specimens

An indirect immunofluorescent antibody procedure (IFA) for the detection and typing of herpes simplex virus (HSV) and an enzyme-linked immunosorbent assay (ELISA) procedure were compared with conventional viral culture. Specimens for culture and ELISA were inoculated into serum free viral transport medium (VTM) and, for IFA, onto slides provided in the kit. Tissue cultures (MRC-5 and primary rabbit kidney) were inoculated and examined daily for cytopathogenic effect (CPE). The remaining VTM was frozen at -70 degrees C until tested by the ELISA system. Slides for IFA were stained with HSV common and HSV-2 specific monoclonal antibodies. Of 155 specimens, 47 (30 percent) were unsatisfactory for the IFA test owing to an inadequate number of epithelial cells on the slides. Of 108 adequate specimens, 45 were culture positive; 39 were positive by the IFA test with a sensitivity of 87 percent and a specificity of 90 percent. Of the 39 positives, 29 (75 percent) were correctly classified as type 1 or type 2, six (15 percent) were typed incorrectly, and four (10 percent) were inadequate for typing by the IFA test. All 155 specimens were suitable for testing by the ELISA procedure. Of 55 specimens positive by culture, only 25 (sensitivity 45 percent) were positive by ELISA. However, the specificity was 100 percent. After incubation of two, three, and six days, the tissue cultures detected 71 percent, 89 percent, and 100 percent of the positives, respectively.     

Sandwich enzyme immunoassay and latex agglutination test for herpes simplex virus keratitis.

We determined the in vitro sensitivities and the diagnostic efficacies of a commercially available latex agglutination test and an enzyme-linked immunosorbent assay using a rabbit model of herpes simplex virus type 1 dendritic keratitis. In contrast to the latex agglutination test, the HERPCHEK enzyme-linked immunosorbent assay offers an extremely sensitive and reliable means of laboratory diagnosis of experimental herpes simplex virus keratitis and is more rapid than viral isolation in tissue culture.