Vol. 29, No. 3
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 650-652
0095-1137/91/030650-03$02.00/0 Copyright © 1991, American Society for Microbiology
Comparison of Standard Culture Methods, a Shell Vial Assay, and a DNA Probe for the Detection of Herpes Simplex Virus LAWTON A. SEAL,lt* PATRICIA S. TOYAMA,' KATHLEEN M. FLEET,' KRAIG S. LERUD,1 SAMUEL R. HETH,2 ANTHONY J. MOORMAN,2 JOSEPH C. WOODS,' AND ROBERT B. HILL' Department of Pathology and Area Laboratory Services' and Department of Obstetrics and Gynecology,2 Tripler Army Medical Center, Honolulu, Hawaii 96859-5000 Received 30 April 1990/Accepted 3 December 1990
A nonradioactive, biotinylated herpes simplex virus (HSV) DNA probe, a shell vial (rabbit kidney cell) culture assay enhanced by a direct fluorescent (HSV monoclonal)-antibody stain at 16 to 20 h postinoculation, and conventional tube cultures with confirmnation via HSV-specific (polyclonal antibody) immunoperoxidase assay were compared for 199 specimens. The predictive values of the positive results were 54.5% for the probe, 95.9% for the shell vial assay, and 100% for the conventional culture methods, while the predictive values of the negative tests were 68.1, 84.0, and 98.4%, respectively. We conclude that the DNA probe (sensitivity, 24.5%; specificity, 88.3%) and the shell vial assay (sensitivity, 66.2%; specificity, 98.4%) cannot be substituted for conventional tube culture techniques (sensitivity, 97.1%; specificity, 100%) in the routine identification of HSV in our laboratory.
imen area of a glass slide. The slide was air dried and fixed in acetone for 10 min at room temperature, and the remainder of the specimen was placed in 2.0 ml of viral transport medium (0.20 M phosphate-buffered sucrose with 50 p.g of gentamicin and 5.0 ,ug of amphotericin B per ml). Next, the viral transport tube and slide were delivered to the laboratory as quickly as possible. Upon arrival, the slide was frozen at -70°C in a desiccator, and the vial was processed for culture. In brief, this consisted of mixing the specimen tube for 30 s with a vortex mixer and removing 1.0 ml of the material for additional antibiotic treatment and low-speed centrifugation (900 x g for 10 min) (2). The resulting supernatant fluid was used as the inoculum for standard tube and shell vial culture assays. Any remaining specimen was stored at -70°C in the viral transport medium pending the need for further analysis (see below). Specimens presented to the laboratory for HSV isolation were cultured routinely in one round-bottom tube of human diploid fibroblasts (MRC-5) and one round-bottom tube of primary rabbit kidney cells (RKC) (Bartels Diagnostics, Baxter Healthcare Corp., West Sacramento, Calif.) by adding 0.25 ml of inoculum to each tube. At the time of inoculation, the ages of the RKC varied from 10 to 25 days, with a mean of 17 days, while the MRC-5 cells were between 8 and 16 days old, with a mean of 13 days. The cultures were incubated for a maximum of 7 days at 37°C in an incubator containing room air. Upon the development of HSV-characteristic cytopathic effects, the cultures were removed from incubation, the monolayer was fixed, and the cells were stained with the Cellmatics herpes simplex virus detection system (Difco Laboratories, Detroit, Mich.) by the laboratory staff according to the manufacturer's directions. The stained tubes were examined under light microscopy at magnifications of x40 and x100 for the presence of greybrown to purple-black HSV-infected cells against an unstained background of normal, uninfected cells. Another 0.25-ml aliquot of processed sample was placed onto the drained (14- to 15-day-old) monolayer of RKC grown on the coverslip at the bottom of a 1-dram (3.7-ml) vial (Bartels Diagnostics). The vial was centrifuged, incubated, and harvested, and the cells were stained in accor-
Substantial advances in the treatment of herpes simplex virus (HSV) infections have been made with the development of antiviral agents, yet the incidence of disease continues to rise (3, 4, 6, 8, 9, 15, 18). Therefore, the need for timely, definitive information with regard to HSV infections still exists, and this need is most critical for near-term women and neonates. However, no reported method of virus detection exceeds standard culture with regard to sensitivity (7). Considerable attention has been given recently to various tests that promise rapid, accurate detection of HSV antigen. Among these are shell vial assays with mono- or polyclonal antibodies used in indirect or direct fluorescentantibody stains, DNA hybridization with various HSVspecific probes, immunoperoxidase staining techniques, latex agglutination assays, and the enzyme-linked immunosorbent assay (ELISA) (1, 4, 5, 11-14, 16, 17, 19-22). We selected three assays for evaluation in our laboratory in a search for a reliable alternative to standard tissue culture assays. These included conventional tube techniques with an immunoperoxidase stain for culture confirmation, centrifugal inoculation of shell vials with a postincubation direct fluorescent-antibody stain by using a monoclonal antibody, and finally a nonradioactive, biotinylated DNA probe for HSV. In our study, no method exceeded routine culture with regard to sensitivity. All 199 samples included in our study were obtained from patients who presented to the Obstetrical and Gynecological Service (Tripler Army Medical Center, Honolulu, Hawaii) with genital lesions having high indexes of suspicion for HSV. The specimens were collected under the supervision of the Obstetrical and Gynecological Service staff (S.R.H., A.J.M.) by standard hospital procedure, which calls for the rupture of vesicles or the removal of crusted material in order to obtain cells from the base of the lesion by abrasion of the area with a cotton-tipped applicator (2, 14). After sampling, the applicator was rolled across the circled spec* Corresponding author. t Present address: Microbiology Branch, Laboratory Sciences Division, Academy of Health Sciences, Fort Sam Houston, TX 78234-6100.
650
VOL. 29, 1991
NOTES
TABLE 1. Comparison of herpes simplex virus identification methodsa
TABLE 2. Assay sensitivity and specificity and predictive values of positive and negative tests
No. of samplesb
Method and result
Predictive value (%) Assay
Sensitivity %)
Specificity (%)
Standard culture +
69 2
0 128
47 24
2 126
18 53
15 113
Shell vial +
DNA probe +
651
a Standard culture versus shell vial culture, P < 0.05; shell vial culture versus DNA probe, P < 0.05; standard culture versus DNA probe, P < 0.01. b As determined by corrected culture results.
dance with manufacturer's directions (Syva Microtrak HSV culture identification test, Syva Company, Palo Alto, Calif.). The slides were viewed by one of the virologists (P.S.T. or L.A.S.) at magnifications of x200 and x400 with a Zeiss Axioskop or Leitz Ortholux microscope fitted with an appropriate light source and filters for fluorescence microscopy. The presence of intracellular, apple-green fluorescence was interpreted as a positive result. The acetone-fixed slides were treated with a nonradioactive, biotinylated DNA probe specific for HSV according to the manufacturer's instructions (Pathogene DNA probe assay; Enzo Diagnostics, New York, N.Y.) by one of the pathologists (K.S.L. or K.M.F.). The completed slides were viewed by standard light microscopy at magnifications of x100 to x400 for the presence of brick-red, intracellular deposits, which are indicative of positive reactivity. Slides with fewer than five intact, appropriately stained epithelial cells were judged inadequate, and these samples were excluded from the study. The results were collected by one of the investigators, recorded, and compared. Attempts were made to reconcile negative cultures with positive results obtained from other assays by reculturing these samples according to the standard methods cited above. This was accomplished usually within 1 to 2 months of the specimen collection date, if adequate samples could be retrieved from storage at -70°C. Statistical analysis of the data included the chi-square distribution and calculations of the sensitivity, specificity, and the predictive values of a positive and a negative test for each method employed. Of the samples that were initially positive by one of the other techniques and reported as culture negative, only two, one each by the DNA probe and the shell vial assay, were determined to be HSV positive by additional culture attempts. We included these samples with those that were culture positive and referred to this set of data as "corrected culture results." In Table 1, these data are compared with the results obtained from the three assay techniques we employed. While standard culture techniques and the shell assay detected 69 and 47 of 71 HSV-positive samples, respectively, the DNA probe assay identified only 18. Chisquare analysis of the data revealed a significant difference in the overall distribution of the results. Results obtained by standard culture differed markedly from those of the shell vial assay (P < 0.05) and differed even more from those of
Standard culture Shell vial with DFA' DNA probe
97.1 66.2 25.4
100 98.4 88.3
Positive test
Negative
100 95.9 54.5
98.4 84.0 68.1
test
a DFA, Direct fluorescent antibody.
the DNA probe (P < 0.01). Likewise, there was considerable variation in the data from the shell vial assay and those obtained with the probe (P < 0.05). The sensitivities, specificities, and the predictive values of positive and negative results for each assay are listed in Table 2. As expected, standard culture techniques were sensitive and specific (97.1 and 100%, respectively), while the shell vial assay had a sensitivity of 66.2% and a specificity of 98.4%. Furthermore, the predictive values of these test results were all greater than 95.0%, with the exception of that calculated for a negative shell vial culture (84.0%). However, the DNA probe assay was not as sensitive (24.5%) or specific (88.3%) as either of the other assays, nor did the predictive values approach those of the culture methods. We calculated the mean time to positive results to be 3.1 days for 234 HSV cultures performed in our laboratory (data not shown). Therefore, the focus of our efforts was to evaluate alternate, more rapid methods of identifying HSV. A test that could produce the same reliable data as routine culture in as few as 2 to 20 h would be of obvious diagnostic value. Unfortunately, neither the shell vial culture nor the DNA probe assay provided the sensitivity that we obtained with standard culture techniques. Tse et al. (20) have reported a sensitivity for the shell vial assays with RKC used to detect HSV greater than that observed by us. In that study, two vials were inoculated with each specimen received; one was stained at 8 h postinfection (sensitivity = 63%) and the other was stained at 20 h (sensitivity = 91%) with the polyclonal immunoperoxidase stain that we employed with our standard culture assay. The results that they obtained from samples stained at 8 h postinoculation closely resembled those we reported after our samples underwent 16 to 20 h of incubation followed by a direct fluorescent-antibody stain with a monoclonal antibody (63 versus 66.2% sensitive, respectively). Both studies used similar sample collection and processing techniques and the same tissue culture cell line, RKC. Fedorko et al. (10) reported an increased sensitivity in the detection of cytomegalovirus when shell vial monolayers that were less than 12 days old were used. Our laboratory must rely on prepared monolayers that are approximately 10 days old when subjected to transoceanic shipment. They require 18 to 24 h to recover from this trauma before use. It is unlikely that we would achieve the level of sensitivity available to laboratories that have tissue culture capabilities. Therefore, it may be reasonable for our laboratory to inoculate two vials for each culture submitted. One would be incubated for the recommended 16- to 20-h period, fixed, and stained accordingly. If a positive result was obtained at that time, no further analysis would be required. However, should this culture test negative, the remaining vial would be available for an additional 8- to 16-h incubation period and subsequent staining. While this two-vial protocol is not suggested by the
652
NOTES
manufacturer for use with the product, it may substantially improve the likelihood of identifying a positive HSV culture within 36 h. In another study (14), the sensitivity and specificity of the DNA probe we employed were reported to be 92 and 63%, respectively. However, the patient population selected for this study comprised individuals who had a history of recurrent HSV infections and who presented with acute episodes of probably recurrent genital herpes. In this highprevalence group, the virus was isolated with much greater frequency (74%) than in our study (35%). While our patients did have lesions with high indexes of suspicion for HSV, a history of recurrent disease was not a criterion for inclusion. (It is unfortunate that no record was kept as to the nature of the lesions [vesicular versus crusted] at the time of sample collection.) Therefore, our population appears to have a lower prevalence of HSV infection than that studied previously (14) with this assay, and it is possible that this DNA probe is less reliable in the detection of HSV in clinical material from a lower-prevalence group. However, in our study, the Pathogene probe performed well as a tissue culture confirmation assay (data not shown). We conclude that standard culture methods are still the most valuable in terms of sensitivity and specificity. We are not convinced, unlike others (14), that the DNA probe assay is a rapid diagnostic tool with utility in our laboratory. Furthermore, our data corroborate those of Woods and Mills (21). They also are unwilling to replace conventional tube cultures with centrifugal inoculation of monolayers followed by overnight incubation and subsequent staining with monoclonal antibodies. We acknowledge that some of the falsepositive results obtained with the shell vial assay or the probe may indeed have been true positives. Our standard tissue culture assay with older commercial cells is unlikely to approach 100% sensitivity. Furthermore, DNA probe positives and conventional culture and shell vial negatives may result from detection of nonviable HSV antigen, while shell vial positives and DNA probe and conventional culture negatives may be due to low viral titers that give rise to random positive results. Sampling error is also a possibility. Further evaluation of these false-positives by electron microscopy or ELISA with neutralization would have been helpful. However, these techniques were beyond the scope of this effort. This work was supported in part by Tripler Army Medical Center Protocols 30H88 and 46H88.
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. Ballew, H. C., H. C. Lyerla, and F. T. Forrester. 1979. Laboratory methods for diagnosing herpesvirus infections, p. 33-65. Centers for Disease Control, Atlanta. 3. Bjorck, L., A. Grubb, and L. Kjellen. 1990. Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus. J. Virol. 64:941-943. 4. Centers for Disease Control. 1986. Annual summary 1986. Morbid. Mortal. Weekly Rep. 33:90. 5. Cleveland, P. H., and D. D. Richman. 1987. Enzyme immunofiltration staining assay for immediate diagnosis of herpes simplex virus and varicella-zoster virus directly from clinical spec-
J. CLIN. MICROBIOL.
imens. J. Clin. Microbiol. 25:416-420. 6. Corey, L. 1984. Genital herpes, p. 449-474. In K. K. Holmes, P.-A. Mardh, P. F. Sparling, and P. J. Wiesner (ed.), Sexually transmitted diseases. McGraw-Hill Book Co., New York. 7. Corey, L. 1986. Laboratory diagnosis of herpes simplex virus infections. Principles guiding the development of rapid diagnostic tests. Diagn. Microbiol. Infect. Dis. 4(Suppl. 3):111-119. 8. Corey, L., H. G. Adams, Z. A. Brown, and K. K. Holmes. 1983. Genital herpes simplex virus infection: clinical manifestations, course and complications. Ann. Intern. Med. 98:958-972. 9. Corey, L., J. Benedetti, C. Critchlow, G. Mertz, J. Douglas, K. Fife, A. Fahnlander, M. Remington, C. Winter, and J. Dargavon. 1983. Treatment of primary first-episode genital herpes infections with acyclovir: results of topical, intravenous and oral therapy. J. Antimicrob. Chemother. 12(Suppl.):79-88. 10. Fedorko, D. P., D. M. Ilstrup, and T. F. Smith. 1989. Effect of age of shell vial monolayers on detection of cytomegalovirus from urine specimens. J. Clin. Microbiol. 27:2107-2109. 11. 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. 12. Hughes, J. H., D. R. Mann, and V. V. Hamparian. 1986. Viral isolation versus immune staining of infected cell cultures for the laboratory diagnosis of herpes simplex virus infections. J. Clin. Microbiol. 24:487-489. 13. Land, S.-A., I. J. Skurrie, and G. L. Gilbert. 1984. Rapid diagnosis of herpes simplex virus infections by enzyme-linked immunosorbent assay. J. Clin. Microbiol. 19:865-869. 14. Langenberg, A., D. Smith, C. L. Brakel, M. Pollice, M. Remington, C. Winter, A. Dunne, and L. Corey. 1988. Detection of herpes simplex virus DNA from genital lesions by in situ hybridization. J. Clin. Microbiol. 26:933-937. 15. Nayyar, K. C., E. Stolz, and M. F. Michel. 1979. Epidemiologic, clinical, diagnostic, and therapeutic aspects. Rising incidence of chancroid in Rotterdam. Br. J. Vener. Dis. 55:439-441. 16. Nerurkar, L. S., M. Namba, G. Brashears, A. J. Jacob, Y. J. Lee, and J. L. Sever. 1984. Rapid detection of herpes simplex virus in clinical specimens by use of a capture biotin-streptavidin enzyme-linked immunosorbent assay. J. Clin. Microbiol. 20:109-114. 17. Prymas, L. A., R. A. Venezia, and M. M. McSharry. 1987. Enhanced method of herpes simplex virus culture confirmation using the Virogen Herpes Slide test. J. Clin. Microbiol. 25:20042005. 18. Reichman, R. C., G. J. Badger, G. J. Mertz, L. Corey, D. D. Richman, J. D. Connor, D. Redfield, M. C. Savoia, M. N. Oxman, Y. Bryson, L. Tyrrell, J. Portnoy, T. Creagh-Kirk, R. E. Keeney, T. Ashikaga, and R. Dolin. 1984. Treatment of recurrent herpes simplex infections with oral acyclovir: a controlled trial. JAMA 251:2103-2108. 19. Salmon, V. C., B. K. Michaels, and R. B. Turner. 1984. Comparison of cell culture with an immunoperoxidase kit for rapid diagnosis of herpes simplex virus infections. Diagn. Microbiol. Infect. Dis. 2:343-345. 20. Tse, P., S. L. Aarnaes, L. M. de la Maza, and E. M. Peterson. 1989. Detection of herpes simplex virus by 8 h in shell vial cultures with primary rabbit kidney cells. J. Clin. Microbiol. 27:199-200. 21. Woods, G. L., and R. D. Mills. 1988. Conventional tube cell culture compared with centrifugal inoculation of MRC-5 cells and staining with monoclonal antibodies for detection of herpes simplex virus in clinical specimens. J. Clin. Microbiol. 26:570572. 22. Ziegler, T., 0. Meurman, P. Arstila, and P. Halomen. 1983. Solid-phase enzyme immunoassay for the detection of herpes simplex virus antigens in clinical specimens. J. Virol. Methods 7:1-9.
JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1991, p. 650-652
0095-1137/91/030650-03$02.00/0 Copyright © 1991, American Society for Microbiology
Comparison of Standard Culture Methods, a Shell Vial Assay, and a DNA Probe for the Detection of Herpes Simplex Virus LAWTON A. SEAL,lt* PATRICIA S. TOYAMA,' KATHLEEN M. FLEET,' KRAIG S. LERUD,1 SAMUEL R. HETH,2 ANTHONY J. MOORMAN,2 JOSEPH C. WOODS,' AND ROBERT B. HILL' Department of Pathology and Area Laboratory Services' and Department of Obstetrics and Gynecology,2 Tripler Army Medical Center, Honolulu, Hawaii 96859-5000 Received 30 April 1990/Accepted 3 December 1990
A nonradioactive, biotinylated herpes simplex virus (HSV) DNA probe, a shell vial (rabbit kidney cell) culture assay enhanced by a direct fluorescent (HSV monoclonal)-antibody stain at 16 to 20 h postinoculation, and conventional tube cultures with confirmnation via HSV-specific (polyclonal antibody) immunoperoxidase assay were compared for 199 specimens. The predictive values of the positive results were 54.5% for the probe, 95.9% for the shell vial assay, and 100% for the conventional culture methods, while the predictive values of the negative tests were 68.1, 84.0, and 98.4%, respectively. We conclude that the DNA probe (sensitivity, 24.5%; specificity, 88.3%) and the shell vial assay (sensitivity, 66.2%; specificity, 98.4%) cannot be substituted for conventional tube culture techniques (sensitivity, 97.1%; specificity, 100%) in the routine identification of HSV in our laboratory.
imen area of a glass slide. The slide was air dried and fixed in acetone for 10 min at room temperature, and the remainder of the specimen was placed in 2.0 ml of viral transport medium (0.20 M phosphate-buffered sucrose with 50 p.g of gentamicin and 5.0 ,ug of amphotericin B per ml). Next, the viral transport tube and slide were delivered to the laboratory as quickly as possible. Upon arrival, the slide was frozen at -70°C in a desiccator, and the vial was processed for culture. In brief, this consisted of mixing the specimen tube for 30 s with a vortex mixer and removing 1.0 ml of the material for additional antibiotic treatment and low-speed centrifugation (900 x g for 10 min) (2). The resulting supernatant fluid was used as the inoculum for standard tube and shell vial culture assays. Any remaining specimen was stored at -70°C in the viral transport medium pending the need for further analysis (see below). Specimens presented to the laboratory for HSV isolation were cultured routinely in one round-bottom tube of human diploid fibroblasts (MRC-5) and one round-bottom tube of primary rabbit kidney cells (RKC) (Bartels Diagnostics, Baxter Healthcare Corp., West Sacramento, Calif.) by adding 0.25 ml of inoculum to each tube. At the time of inoculation, the ages of the RKC varied from 10 to 25 days, with a mean of 17 days, while the MRC-5 cells were between 8 and 16 days old, with a mean of 13 days. The cultures were incubated for a maximum of 7 days at 37°C in an incubator containing room air. Upon the development of HSV-characteristic cytopathic effects, the cultures were removed from incubation, the monolayer was fixed, and the cells were stained with the Cellmatics herpes simplex virus detection system (Difco Laboratories, Detroit, Mich.) by the laboratory staff according to the manufacturer's directions. The stained tubes were examined under light microscopy at magnifications of x40 and x100 for the presence of greybrown to purple-black HSV-infected cells against an unstained background of normal, uninfected cells. Another 0.25-ml aliquot of processed sample was placed onto the drained (14- to 15-day-old) monolayer of RKC grown on the coverslip at the bottom of a 1-dram (3.7-ml) vial (Bartels Diagnostics). The vial was centrifuged, incubated, and harvested, and the cells were stained in accor-
Substantial advances in the treatment of herpes simplex virus (HSV) infections have been made with the development of antiviral agents, yet the incidence of disease continues to rise (3, 4, 6, 8, 9, 15, 18). Therefore, the need for timely, definitive information with regard to HSV infections still exists, and this need is most critical for near-term women and neonates. However, no reported method of virus detection exceeds standard culture with regard to sensitivity (7). Considerable attention has been given recently to various tests that promise rapid, accurate detection of HSV antigen. Among these are shell vial assays with mono- or polyclonal antibodies used in indirect or direct fluorescentantibody stains, DNA hybridization with various HSVspecific probes, immunoperoxidase staining techniques, latex agglutination assays, and the enzyme-linked immunosorbent assay (ELISA) (1, 4, 5, 11-14, 16, 17, 19-22). We selected three assays for evaluation in our laboratory in a search for a reliable alternative to standard tissue culture assays. These included conventional tube techniques with an immunoperoxidase stain for culture confirmation, centrifugal inoculation of shell vials with a postincubation direct fluorescent-antibody stain by using a monoclonal antibody, and finally a nonradioactive, biotinylated DNA probe for HSV. In our study, no method exceeded routine culture with regard to sensitivity. All 199 samples included in our study were obtained from patients who presented to the Obstetrical and Gynecological Service (Tripler Army Medical Center, Honolulu, Hawaii) with genital lesions having high indexes of suspicion for HSV. The specimens were collected under the supervision of the Obstetrical and Gynecological Service staff (S.R.H., A.J.M.) by standard hospital procedure, which calls for the rupture of vesicles or the removal of crusted material in order to obtain cells from the base of the lesion by abrasion of the area with a cotton-tipped applicator (2, 14). After sampling, the applicator was rolled across the circled spec* Corresponding author. t Present address: Microbiology Branch, Laboratory Sciences Division, Academy of Health Sciences, Fort Sam Houston, TX 78234-6100.
650
VOL. 29, 1991
NOTES
TABLE 1. Comparison of herpes simplex virus identification methodsa
TABLE 2. Assay sensitivity and specificity and predictive values of positive and negative tests
No. of samplesb
Method and result
Predictive value (%) Assay
Sensitivity %)
Specificity (%)
Standard culture +
69 2
0 128
47 24
2 126
18 53
15 113
Shell vial +
DNA probe +
651
a Standard culture versus shell vial culture, P < 0.05; shell vial culture versus DNA probe, P < 0.05; standard culture versus DNA probe, P < 0.01. b As determined by corrected culture results.
dance with manufacturer's directions (Syva Microtrak HSV culture identification test, Syva Company, Palo Alto, Calif.). The slides were viewed by one of the virologists (P.S.T. or L.A.S.) at magnifications of x200 and x400 with a Zeiss Axioskop or Leitz Ortholux microscope fitted with an appropriate light source and filters for fluorescence microscopy. The presence of intracellular, apple-green fluorescence was interpreted as a positive result. The acetone-fixed slides were treated with a nonradioactive, biotinylated DNA probe specific for HSV according to the manufacturer's instructions (Pathogene DNA probe assay; Enzo Diagnostics, New York, N.Y.) by one of the pathologists (K.S.L. or K.M.F.). The completed slides were viewed by standard light microscopy at magnifications of x100 to x400 for the presence of brick-red, intracellular deposits, which are indicative of positive reactivity. Slides with fewer than five intact, appropriately stained epithelial cells were judged inadequate, and these samples were excluded from the study. The results were collected by one of the investigators, recorded, and compared. Attempts were made to reconcile negative cultures with positive results obtained from other assays by reculturing these samples according to the standard methods cited above. This was accomplished usually within 1 to 2 months of the specimen collection date, if adequate samples could be retrieved from storage at -70°C. Statistical analysis of the data included the chi-square distribution and calculations of the sensitivity, specificity, and the predictive values of a positive and a negative test for each method employed. Of the samples that were initially positive by one of the other techniques and reported as culture negative, only two, one each by the DNA probe and the shell vial assay, were determined to be HSV positive by additional culture attempts. We included these samples with those that were culture positive and referred to this set of data as "corrected culture results." In Table 1, these data are compared with the results obtained from the three assay techniques we employed. While standard culture techniques and the shell assay detected 69 and 47 of 71 HSV-positive samples, respectively, the DNA probe assay identified only 18. Chisquare analysis of the data revealed a significant difference in the overall distribution of the results. Results obtained by standard culture differed markedly from those of the shell vial assay (P < 0.05) and differed even more from those of
Standard culture Shell vial with DFA' DNA probe
97.1 66.2 25.4
100 98.4 88.3
Positive test
Negative
100 95.9 54.5
98.4 84.0 68.1
test
a DFA, Direct fluorescent antibody.
the DNA probe (P < 0.01). Likewise, there was considerable variation in the data from the shell vial assay and those obtained with the probe (P < 0.05). The sensitivities, specificities, and the predictive values of positive and negative results for each assay are listed in Table 2. As expected, standard culture techniques were sensitive and specific (97.1 and 100%, respectively), while the shell vial assay had a sensitivity of 66.2% and a specificity of 98.4%. Furthermore, the predictive values of these test results were all greater than 95.0%, with the exception of that calculated for a negative shell vial culture (84.0%). However, the DNA probe assay was not as sensitive (24.5%) or specific (88.3%) as either of the other assays, nor did the predictive values approach those of the culture methods. We calculated the mean time to positive results to be 3.1 days for 234 HSV cultures performed in our laboratory (data not shown). Therefore, the focus of our efforts was to evaluate alternate, more rapid methods of identifying HSV. A test that could produce the same reliable data as routine culture in as few as 2 to 20 h would be of obvious diagnostic value. Unfortunately, neither the shell vial culture nor the DNA probe assay provided the sensitivity that we obtained with standard culture techniques. Tse et al. (20) have reported a sensitivity for the shell vial assays with RKC used to detect HSV greater than that observed by us. In that study, two vials were inoculated with each specimen received; one was stained at 8 h postinfection (sensitivity = 63%) and the other was stained at 20 h (sensitivity = 91%) with the polyclonal immunoperoxidase stain that we employed with our standard culture assay. The results that they obtained from samples stained at 8 h postinoculation closely resembled those we reported after our samples underwent 16 to 20 h of incubation followed by a direct fluorescent-antibody stain with a monoclonal antibody (63 versus 66.2% sensitive, respectively). Both studies used similar sample collection and processing techniques and the same tissue culture cell line, RKC. Fedorko et al. (10) reported an increased sensitivity in the detection of cytomegalovirus when shell vial monolayers that were less than 12 days old were used. Our laboratory must rely on prepared monolayers that are approximately 10 days old when subjected to transoceanic shipment. They require 18 to 24 h to recover from this trauma before use. It is unlikely that we would achieve the level of sensitivity available to laboratories that have tissue culture capabilities. Therefore, it may be reasonable for our laboratory to inoculate two vials for each culture submitted. One would be incubated for the recommended 16- to 20-h period, fixed, and stained accordingly. If a positive result was obtained at that time, no further analysis would be required. However, should this culture test negative, the remaining vial would be available for an additional 8- to 16-h incubation period and subsequent staining. While this two-vial protocol is not suggested by the
652
NOTES
manufacturer for use with the product, it may substantially improve the likelihood of identifying a positive HSV culture within 36 h. In another study (14), the sensitivity and specificity of the DNA probe we employed were reported to be 92 and 63%, respectively. However, the patient population selected for this study comprised individuals who had a history of recurrent HSV infections and who presented with acute episodes of probably recurrent genital herpes. In this highprevalence group, the virus was isolated with much greater frequency (74%) than in our study (35%). While our patients did have lesions with high indexes of suspicion for HSV, a history of recurrent disease was not a criterion for inclusion. (It is unfortunate that no record was kept as to the nature of the lesions [vesicular versus crusted] at the time of sample collection.) Therefore, our population appears to have a lower prevalence of HSV infection than that studied previously (14) with this assay, and it is possible that this DNA probe is less reliable in the detection of HSV in clinical material from a lower-prevalence group. However, in our study, the Pathogene probe performed well as a tissue culture confirmation assay (data not shown). We conclude that standard culture methods are still the most valuable in terms of sensitivity and specificity. We are not convinced, unlike others (14), that the DNA probe assay is a rapid diagnostic tool with utility in our laboratory. Furthermore, our data corroborate those of Woods and Mills (21). They also are unwilling to replace conventional tube cultures with centrifugal inoculation of monolayers followed by overnight incubation and subsequent staining with monoclonal antibodies. We acknowledge that some of the falsepositive results obtained with the shell vial assay or the probe may indeed have been true positives. Our standard tissue culture assay with older commercial cells is unlikely to approach 100% sensitivity. Furthermore, DNA probe positives and conventional culture and shell vial negatives may result from detection of nonviable HSV antigen, while shell vial positives and DNA probe and conventional culture negatives may be due to low viral titers that give rise to random positive results. Sampling error is also a possibility. Further evaluation of these false-positives by electron microscopy or ELISA with neutralization would have been helpful. However, these techniques were beyond the scope of this effort. This work was supported in part by Tripler Army Medical Center Protocols 30H88 and 46H88.
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