Gingivostomatitis is the most common clinical manifestation of primary herpes simplex virus (HSV) infection in young children. The typical oral and extraoral lesions make the diagnosis straightforward in most cases, although the differential diagnosis could include herpangina, aphthous stomatitis and Stevens-Johnson syndrome. Rapid and accurate diagnosis of HSV gingivostomatitis can be important in the prevention of outbreaks in closed communities by isolation of infected children or by the administration of acyclovir as a prophylaxis1 and for early treatment with acyclovir when clinical symptoms and signs are inconclusive. A rapid monoclonal antibody-based enzyme immunoassay (EIA) (SureCell®; Kodak, Rochester, NY) for the direct detection of HSV antigen types 1 and 2 from clinical specimens has recently been evaluated in genital herpes. Sensitivity ranged from 64.4 to 81.1%, with 100% specificity.2, 3 When it was used for ocular HSV infection, a low sensitivity of 27% and a specificity of 80% were reported.4 In this work we compared this rapid EIA in young children with early gingivostomatitis with virus isolation in tissue culture, presently accepted as the standard diagnostic test. Subjects and methods. Children with clinical manifestations of gingivostomatitis present for fewer than 72 h were identified by their primary pediatrician and referred to the investigation center. The minimal criteria for diagnosis were ulcerating vesicles on the oral mucosa and tongue accompanied by systemic fever (≥38°C). In more advanced cases swollen gums, ulcerating gums that bled easily and extraoral vesicular lesions were present. Parents of all children participating in the study gave their informed consent. A specimen was collected from each child using a double Dacron®-tipped swab which was gently rubbed over the oral lesions. Blood samples for HSV serologic assay were obtained at the time of evaluation and 14 ± 2 days later. HSV culture. One swab was placed in 2 ml of transport medium.5 The specimens were either sent immediately over ice or kept refrigerated overnight and sent the next day to the Central Virology Laboratory, Tel Hashomer, Israel. The swabs were agitated and inoculated into tubes containing monolayers of human embryo fibroblasts, Vero cells and Buffalo green monkey kidney cells prepared with 10-5 M dexamethasone.6 After incubation at 37°C for 24 h the medium was replaced with fresh Eagle's minimal essential medium supplemented with nonessential amino acids containing 2% fetal calf serum. The cultures were examined for cytopathic effects. When 50% of the cells showed a cytopathic effect the supernatant was stored, and the cells were harvested, washed and used to prepare slides for immunofluorescence staining. Serotyping of HSV with monoclonal antibodies. All isolates of HSV were typed by fluorescence conjugate monoclonal antibodies (Syva Microtak HSV-1®, HSV-2®; Palo Alto, CA), according to the manufacturer's instructions. Infected Buffalo green monkey cells on slides, air dried and fixed in acetone were exposed to monoclonal antibodies to HSV-1 or HSV-2 for 15 min at 37°C, rinsed with distilled water, air dried and mounted. The slides were examined with a Zeiss fluorescence microscope equipped with an MBO mercury lamp. Each test included known positive and negative controls. Serology assays. An indirect immunofluorescence antibody (IFA) test was performed as described previously.7 In brief baby hamster kidney cells were inoculated with HSV-1 (VR 3 strain) after overnight incubation at 36°C. Drops of twicewashed cell suspension containing 1 × 106 cells/ml were placed on glass slides, dried, fixed for 1 min in cold acetone and used after storage at -20°C. Twofold dilutions of serum (ranging from 1:8 to 1:512) were added to the HSV-infected and the uninfected baby hamster kidney cells. After incubation for 35 min at 36°C and washing for 15 min, the baby hamster kidney cells were treated for 30 min with fluorescein-conjugated anti-human IgG prepared and standardized in our laboratory. After another 15-min wash slides were mounted in 10% glycerolphosphate-buffered saline and examined at a magnification of ×40 with a Zeiss fluorescence microscope equipped with an HBO 200 mercury lamp. Positive and negative sera were included as controls. The antibody titer was expressed as the reciprocal of the last dilution giving distinct specific fluorescence in the infected cells. Rapid EIA assay. The Kodak SureCell Herpes Kit® was used to detect HSV antigen directly from swabs. The second Dacron-tipped swab was placed within 30 min of collection into flexible plastic tubes containing 1.0 ml of extraction buffer. The swab was rotated and squeezed for 1 min, after which the solution was squeezed through a filter into three tests wells (The tubes and filters are part of the test kit.), a negative control well, a sample well and a positive control well that contained both HSV-1 and HSV-2 antigens. The solution drained through the test wells and any HSV antigen was bound to the filter membrane at the bottom of the well. After the wells were rinsed and subjected to hydrogen peroxide treatment, an antibody conjugate specific for HSV-1 and HSV-2 was added to the sample and positive control wells, and a HSV nonspecific one was added to the negative control well. The wells were incubated for 5 min at room temperature and rinsed twice. Leuco dye was then added and the color in the wells assessed after 5 min; the negative control should remain white and the positive control should turn a uniform red-pink. The sample well finding was interpreted by semi-quantitiative scoring: 3+, the sample well was as red-pink as the positive control; 2+, the sample well was clearly red-pink, but less so than the positive control; 1+, the sample well was hardly pink and was compared with the negative control while covering the positive control. A positive result was recorded when the sample well was uniformly more pink than the negative control. Results. Eighty children (41 female, 39 male) of ages ranging from 10 to 72 months were included in the study. Positive HSV cultures were found in 67 (83.8%). All positive HSV cultures were identified as HSV-1. Positive EIA results were detected in 64 samples, with the following intensity scoring: 3+, 13 (20.3%); 2+, 42 (65.6%); 1+, 9 (14.1%). Of the 67 samples with positive HSV cultures 64 yielded positive rapid EIA results including all ranges of intensity (1+ through 3+), for a sensitivity of 95.5% (Table 1). The EIA test was negative in all negative HSV cultures for a specificity of 100%. The EIA results were in agreement with the cultures in 96.3% of cases. The positive and negative predictive values were 100 and 81.3%, respectively. When the results were recalculated by including only the easily read reactions of 2+ and 3+ as a positive EIA, the sensitivity, specificity, positive predictive value, negative predictive value and agreement were 82.1, 100, 100, 52 and 85%, respectively (Table 1). Acute and convalescent serum samples were available for serologic HSV study in 54 children and convalescent samples only in another 3. All 13 samples of the children with negative HSV cultures had a negative IFA titer of ≥4 in the convalescent sample. Of the 44 children with positive HSV results, 41 cases showed a 4-fold increase in IFA titer, with a convalescence titer of ≥64; the remaining 3 had IFA titers of ≥64 in a single convalescent sample. Discussion. The efficacy of the Kodak SureCell Herpes Kit® was evaluated in young children with suspected herpetic gingivostomatitis. HSV type 1 was identified in 83.8% of these children by isolation on tissue culture, which was in good agreement with the serologic studies. The sensitivity of the EIA was 95.5% and the specificity was 100%. These good results were achieved when the rapid test was performed carefully, and the negative results were determined only after comparison of the color of the sample well with the negative control. We found that some samples that appeared to be negative at first glance were different from the negative control when reexamined by covering the positive control well, which was usually deep red. This type of borderline result was detected in 14.1% of the children with a positive EIA assay. We assume that these specimens had a lower virus titer than the specimens with the intense color reaction, as shown previously by false negative results.2 Earlier studies with the same EIA in adult patients with suspected urogenital herpes infection2, 3 reported sensitivities of 64.4 and 81.1% and high specificities of 98.9 and 100% in the two studies. The studies used different culture methodologies and the assay results were also influenced by the type of specimen submitted (vesicle, nonvesicle) and the source (male, female).2, 3 Disappointing results with the Kodak SureCell® EIA have been reported in ocular infection with HSV (sensitivity 27%, specificity 80%).4 A low sensitivity of only 50% was also reported in a small subgroup of six patients with herpetic oral-throat lesions.2 The present good results with the EIA test in herpetic gingivostomatitis could be attributable to several factors: (1) relatively high inocula because the oral lesions obtained from the children were fresh, ranged in number from 5 to >20 and were easily collected; (2) all children were sick for fewer than 72 h, probably without circulating antibodies which can interfere with antigen detection; (3) high rate of HSV infection among the study group as determined by cell culture (83.8%); (4) early performance of the test, within 30 min after collection of the specimen; (5) careful evaluation of the weak equivocal results by experienced examiners. In addition the fact that HSV-1 was isolated in all cases might also have affected the results. In a recent review of the various tests for detecting HSV infections,8 the EIA was recommended as a useful, convenient and reliable technique. The polymerase chain reaction9 for rapid detection of HSV in saliva of patients with gingivostomatitis was also reliable, but it is unsuitable for clinical settings such as the medical office or emergency room because the technical procedures require trained personnel and special equipment. Kodak's rapid EIA kit is convenient and needs no special equipment or training with results available in fewer than 15 min. Although the maximal sensitivity in this study was 95.5%, in routine clinical examinations, the lower sensitivity of 82.1%, as calculated from the data that considered also the 1 + (weak) positive results as negative is more realistic. The specificity was found by us and others2, 3 to be very high. The use of this rapid test for in making a decision of early treatment of herpetic gingivostomatitis could save those with nonherpetic gingivostomatis (16.3% of the children in the present population) from unnecessary medication. Routine cell line culture are recommended in patients with clinically suspected herpetic gingivostomatitis and a negative rapid EIA. Jacob Amir, M.D.; Rachel Straussberg, M.D.; Liora Harel, M.D.; Zehava Smetana, M.D.; Itzhak Varsano, M.D. Department of Pediatrics C Schneider Children's Medical Center of Israel Petah Tiqva, Israel (JA, RS, LH, IV) Sackler School of Medicine Tel Aviv University, Tel Aviv, Israel (JA, RS, LH, IV) Central Virology Laboratory Chaim Sheba Medical Center Tel Hashomer, Israel (ZS)
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Amir et al. (1996) studied this question.