JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1992, P. 581-5840095-1137/92/030581-04$02.00/0Copyright © 1992, American Society for Microbiology

Vol. 30, No. 3

Detection of Herpes Simplex Virus by a NonradiometricSpin-Amplified In Situ Hybridization AssayMICHAEL S. FORMAN,* CINDY S. MERZ, AND PATRICIA CHARACHE

Department ofLaboratory Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21205

Received 28 August 1991/Accepted 23 November 1991

An in situ hybridization kit (Diagnostic Hybrids, Inc., Athens, Ohio) was evaluated for use in the detectionand identification of herpes simplex virus (HSV) from clinical specimens. For in situ hybridization, a 10-minspin amplification onto monolayers of African green monkey kidney cells (CV-1) in 24-well polystyrene dishes,24-h culture amplification, and hybridization with an alkaline phosphatase-labeled DNA probe were used. Atotal of 648 specimens were tested, including 275 specimens from patients with symptomatic diseases sentspecifically for HSV detection and 373 specimens from asymptomatic immunocompromised patients sent fordetection of HSV shedding. Overall, the sensitivity of the hybridization assay was 97.8% (131 of 134specimens), with 105 of 105 (100%l ) specimens from symptomatic patients and 26 of 29 (89.9%o) specimens fromasymptomatic patients being detected. The three specimens that were false negative by in situ hybridization hadlow virus titers, as determined by tissue culture. The specificity was 99.6% (512 of 514 specimens). The rapid,accurate results suggest that the in situ hybridization kit may be used as an alternative to conventional tissueculture for the detection of HSV.

Herpes simplex virus (HSV) causes a variety of diseasesin humans, ranging from common localized diseases, such asherpes labialis, aphthous stomatitis, keratoconjunctivities,and infections of the genital tract, to rare but life-threateninginfections such as meningoencephalitis, pneumonia, andsystemic neonatal infection. Accurate, timely diagnosis ofHSV is important, since effective anti-HSV agents are nowavailable for treatment of these diseases. In addition, rapiddetection of HSV shedding in immunocompromised patients(2, 9, 13) can be important for monitoring the effectiveness ofprophylaxis and for identifying the development of acyclovirresistance (5).A number of technologies have been applied for the

detection and identification of HSV. In addition to conven-tional tissue culture (1), these include latex agglutination (6),immunofluorescence (7), immunoperoxidase staining (8),enzyme-linked immunosorbent assay (10), and nucleic acidhybridization with and without nucleic acid amplification(11, 12). Each approach has its advantages and limitations interms of its relative sensitivity, specificity, ease of perfor-mance, equipment requirements, and speed of detection.

In this study we evaluated an in situ hybridization (ISH)assay that uses an alkaline phosphatase enzyme-labeledHSV-specific DNA probe for detection and identification ofHSV in cell culture after 24 h of amplification. This assaywas compared with a tissue culture reference method (TC-RM). Specimens from patients with active, symptomaticHSV infections and specimens from asymptomatic, immu-nocompromised patients, which were obtained to determineHSV shedding, were tested.

MATERIALS AND METHODSSpecimens tested. A total of 648 clinical specimens were

analyzed, including 210 specimens from active genital le-sions, 391 specimens from upper respiratory sites, and 47specimens from other diverse anatomic sites, including skin,conjunctiva, perianal ulcers, and esophageal ulcers. Two

* Corresponding author.

subgroups of specimens were analyzed separately. Group Ispecimens were from symptomatic patients suspected ofhaving active HSV infection. These included 89 specimensfrom obstetrical or gynecological patients, 106 specimensfrom patients with sexually transmitted diseases, 19 speci-mens from patients with AIDS, and 61 specimens from othermiscellaneous patient populations. Group II specimens werefrom immunocompromised patients without active lesions.It included 94 specimens from patients with aplastic leuke-mia, 176 specimens from bone marrow transplant recipients,97 specimens from other oncology patients, and 6 specimensfrom organ transplant recipients (liver, kidney, and heart).These specimens were also tested for other viral pathogensincluding cytomegalovirus, adenovirus, and parainfluenzaviruses, in addition to HSV.

Specimens from active lesions were collected with dacronswabs, and those from asymptomatic patients were collectedwith either throat swabs or saline gargles. All specimens senton swabs were transported to the laboratory in transportmedium (minimum essential medium with 5% fetal bovineserum, 50 ,ug of gentamicin per ml, and 5 ,ug of amphotericinper ml). Gargles were collected in sterile containers andwere immediately transported to the laboratory.TC-RM. Group I specimens were inoculated onto each of

the following three cell lines: MCR-5, human neonatal kid-ney, and primary rabbit kidney. Group II specimens wereinoculated onto each of the following four cell lines: Wi-38,MRC-5, human neonatal kidney, and cynomologus monkeykidney. A 0.2-ml aliquot of specimen was inoculated intoeach cell line in culture tubes (16 by 125 mm; WhittakerBioproducts, Walkersville, Md.). The tubes were rotated for90 min for virus absorption, washed once with Hanksbalanced salt solution, fed with 1.5 ml of minimum essentialmedium with 2% fetal bovine serum, and incubated at 370Cin 5% CO2. All HSV cultures from group I specimens wereobserved daily for 7 days. Group II specimens, which wereprocessed for other viral pathogens as well as for HSV, wereobserved for 21 days. When cytopathic effect (CPE) consis-tent with HSV was noted, cells were scraped from the tube,washed with phosphate-buffered saline, and spotted onto

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FIG. 1. Appearance of HSV foci in African green monkey kidney cells (CV-1) detected by the nonradiolabeled ISH assay. (A) Stockstrain, HSV strain 1F. (B) Clinical isolate of HSV. Magnifications, x 100.

microscope slides. They were air dried, fixed in cold ace-tone, stained with HSV type-specific monoclonal immuno-fluorescent antibodies (Syva, Palo Alto, Calif.), and scoredfor total number of cells, the number and intensity offluorescing cells, and HSV type (type 1 or 2).ISH method. The ISH kit (Diagnostic Hybrids, Inc.,

Athens, Ohio) included 24-well culture dishes containingAfrican green monkey kidney cells (CV-1), replacementmedium (Eagle minimum essential medium with 5% fetalbovine serum), blocking solution, fixative, wash concentratesolution, and probe hybridization solution containing a sin-gle-stranded, HSV-specific DNA probe chemically linked toalkaline phosphatase in a buffered aqueous solution with25% formamide. The probe is a 2-kb fragment that includes800- and 1,200-bp PstI discontiguous regions of DNA fromHSV strain 1F that are cloned in bacteriophage M13mpl9 asdescribed previously (15). It is reactive with both HSV type1 (HSV-1) and HSV-2 (14). Positive and negative controlswere not provided.Medium was aspirated from the wells, and 1.0 ml of

replacement medium was added. The patient specimen (0.2ml) was then inoculated into duplicate wells. A positivecontrol, HSV strain 1F at 1.5 x 10 PFU/ml (American TypeCulture Collection Rockville, Md.), and a negative control(viral transport medium) were included on each plate tested.Plates were centrifuged (700 x g) for 10 min at room

temperature and were then incubated at 37°C in 5% CO2.At 24 h, the medium was aspirated from each of the 24

wells, and the plates were submerged in 95% ethyl alcoholfor 5 min. Plates were drained for 10 s, and 0.25 ml ofblocking solution was added for 5 min at room temperatureto reduce nonspecific background staining. The monolayerswere then fixed, and DNA was denatured by the addition of0.25 ml of fixative solution containing sodium hydroxide for5 min at room temperature. Then, 0.25 ml of probe hybrid-

ization solution containing alkaline phosphatase-labeledHSV probe was added. The plates were incubated in a 45°Cwater bath for 30 min. The probe solution was aspirated,each well was washed four times, 0.5 ml of wash solutionwas added, and then the plates were allowed to incubate foran additional 10 min in a 45°C water bath. After one

additional wash, 0.25 ml of an aqueous chromogenic sub-strate solution containing 50% dimethylformamide, bromo-chyloroindolyl phosphate, and nitroblue tetrazolium dye wasadded to each well; plates were again incubated in a 45°Cwater bath for 1 h. The plates were washed with water andexamined with an inverted light microscope at x 40 and x 100magnification. The presence of a purple stain in the nucleusof the CV-1 cells defined a positive test result.

RESULTS

ISH staining characteristics. The staining pattern for theHSV strain 1F control was distinctly different from that ofthe clinical isolates (Fig. 1). HSV 1F foci remained intact,with an average of 20 infected cells per focus. Clinicalisolates were lytic, showing greater cell destruction anddetachment, often with positive cells remaining only at theperiphery of the focus. This, however, presented no inter-pretive problem, even in specimens with high virus titers; ineach case, enough stained cells remained fixed to the wells topermit easy, accurate reading. The backgrounds of clinicalspecimens with large numbers of epithelial cells stainedbrown, which was easily distinguished from positive cells,which had purple nuclei.

Sensitivity of ISH assay. HSV strain 1F adjusted to a titerof 1.0 x 103 PFU/ml was serially diluted; 0.2 ml of eachdilution was inoculated onto each of two hybridization wells

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DETECTION OF HSV BY HYBRIDIZATION 583

TABLE 1. Assessment of sensitivity of the ISH assay versus thatof conventional tissue culture

HSV strain 1F Detection of HSV CPE in tissueinoculum ISH result culture on day:

(PFU/0.2 ml) at 24 h 1 2 3 4

200 + + + + +100 + + + + +50 + + + + +25 + + + + +12.5 + + + + +6.25 + 0 + + +3.12 + 0 + + +1.56 + 0 0 + +0.78 0 0 0 0 +

and into one tube each of MRC-5 and primary rabbit kidneycell monolayers. Wells for ISH were processed at 24 h asdescribed above, whereas TC-RMs were read for CPE dailyfor 7 days. The effect of the HSV IF inoculum size on theresults of the ISH assay versus those of TC-RM was used asan assessment of sensitivity (Table 1). Because the HSV 1Fcontrol causes less cell lysis than clinical isolates, it isdifficult to detect early CPE in tissue culture. By day 3, thereference method attained a level of sensitivity equal to thatof the 24-h ISH assay; by day 4, with an inoculum repre-senting less than 1 PFU of HSV, TC-RM showed greatersensitivity. The in vitro sensitivity of the ISH assay wasestimated to be 8 PFU/ml.

Detection of HSV in clinical specimens. A total of 648specimens submitted to the virology laboratory, either spe-cifically for culture of HSV from specimens from sympto-matic patients (group I) or for surveillance of HSV sheddingfrom asymptomatic patients (group II), were analyzed bothby the 24-h ISH method and by TC-RM (Table 2). Overall,134 (20.7%) specimens were positive by the referencemethod. Positive specimens included 50 isolates of HSV-1and 84 isolates of HSV-2. By the ISH assay, 133 (20.5%)specimens were positive. Although the ISH procedure doesnot include screening for CPE, 20 of 133 ISH-positivespecimens had no visible signs of CPE and appeared to be

TABLE 2. Detection of HSV by ISH and conventionaltissue culture

No. of No. (%) positive by:Group and anatomic spcimenssite tested ISH Reference

Group I (symptomatic patients)Genital 210 87 (41.4) 85 (40.4)Skin 24 11 (45.8) 11 (45.8)Orofacial 18 4 (22.2) 4 (22.2)Conjunctiva 10 2 (20.0) 2 (20.0)Other' 13 3 (23.1) 3 (23.1)Subtotal 275 107 (38.9) 105 (38.2)

Group II (asymptomatic patients), 373b 26 (7.0) 29 (7.8)Throat

Total 648 133 (20.5) 134 (20.7)a Specimens were from rectum (n = 6), esophagus (n = 5), umbilicus (n =

1), and blood (n = 1).b Three specimens grew cytomegalovirus and one specimen grew respira-

tory syncytial virus. These four specimens were negative for HSV by the ISHassay.

TABLE 3. Analysis of ISH assay performance

Statistical values (%)

Group I Group II Total

Sensitivity 105/105 (100) 26/29 (89.9) 131/134 (97.8)Specificity 168/170 (98.8) 344/344 (100) 512/514 (99.6)Positive predictive 105/107 (98.1) 26/26 (100) 131/133 (98.5)value

Negative predic- 170/170 (100) 344/347 (99.1) 514/517 (99.4)tive value

similar to the staining pattern of HSV strain 1F, as shown inFig. 1A.The performance of the ISH assay in group I specimens

was evaluated separately. As expected, the population fromwhich these specimens were obtained showed the highestrecovery rate of HSV (38.2%). The hybridization assaydetected HSV in 107 specimens, 2 more than were found tobe HSV positive by TC-RM. The two additional specimenswere genital specimens, each of which produced a singlefocus of stained cells in one of the two hybridization wells.The ISH assay was further analyzed for the detection of

HSV shedding by using group II specimens. A recovery rateof 7.8% was observed by TC-RM whereas the hybridizationassay had a recovery rate of 7%. The three specimens thatwere negative by ISH each showed CPE in only one of threetissue culture tubes after 6, 7, or 8 days of incubation.

Overall, when times to detection were compared, HSVwas detected more rapidly in the ISH assay than it was in theTC-RM. Final results were available in 24 h with the ISHassay, while TC-RM required between 1 and 8 days (mean,1.88 days). The time to detection was 1 day for the ISH assayversus a mean of 1.57 days with group I specimens and 2.76days with group II specimens. Even after the initial detectionof CPE by TC-RM, an additional day was frequently re-quired to amplify the number of infected cells and permitconfirmation by immunofluorescence, while further proce-dures were not required by the ISH assay.Data analysis of the ISH assay results is presented in

Table 3. The ISH assay was sensitive (97.8%) and specific(99.6%) for the detection of HSV. Detection ofHSV in groupII specimens by the ISH assay was slightly less sensitive,probably because of the low levels of virus shed in thatpatient population. Positive and negative predictive valuesexceeded 98% in both groups of patient specimens.

DISCUSSION

The ISH assay presents an alternative to the conventionaldetection of HSV by tissue culture. It combines the sensi-tivity of cell culture isolation with the specificity of DNAhybridization. Unlike TC-RM, which required the subjectivedetection of HSV CPE and a subsequent confirmationmethod, the ISH assay permits the simultaneous detectionand identification of HSV. The most significant advantage ofthe ISH assay is its rapid turnaround time. Positive andnegative specimens are reported concurrently, allowing forappropriate patient management that would include thetimely initiation of antiviral therapy for HSV-positive pa-tients and further diagnostic procedures, as required, forHSV-negative ones. The rapid, sensitive detection of HSVdemonstrated in the present study is similar to rapid antigendetection in shell vial cultures (3). This is probably due to thespin amplification step used in both methods and to the

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detection of viral DNA prior to the development of CPE intissue culture. The ISH assay was comparable to TC-RM inits ability to detect HSV in specimens from a wide variety ofbody sites and clinical situations and was highly specific forHSV-1 and HSV-2. Cross-reactivity of the probe against awide range of both DNA and RNA viruses has been deter-mined previously (4). In this study, one isolate of respiratorysyncytial virus and three of cytomegalovirus from group IIpatient specimens were recovered in tissue culture and didnot cross-react with the HSV hybridization probe.The five discrepant results between ISH and TC-RM were

reviewed. Both specimens positive by the ISH assay andnegative by TC-RM were genital specimens from sympto-matic patients (group I specimens). Other specimens fromthese patients were not available, and reculturing of a frozenaliquot again gave negative results, although a review of thepatients' charts supported a clinical diagnosis of genital HSVinfection. The three group II specimens that had delayedpositivity by TC-RM and that were negative by ISH wereconsidered false-negative ISH assay results. A review of thepatients' charts supports this conclusion, since additionalthroat specimens from each of the three patients werepositive for HSV. The inability of ISH to detect viralshedding in these cases appears to reflect the limitation insensitivity that parallels the results of the sensitivity assess-ment (Table 1) with dilution of stock viruses. In these threecases, the virus took 6, 7, and 8 days postinoculation beforeCPE appeared, which is evidence that virus was present in alow titer in these surveillance specimens.The hybridization assay was easy to perform. All reagents

and supplies, except controls, are provided in the ISH kit.Multiple samples can be readily processed concurrently onthe same plate. Although the various test steps, includingwashes, incubations, and reagent additions, were repetitive,they were not complicated and could be performed easilyand rapidly. Daily readings of tissue culture tubes could beeliminated. Wells were easy to interpret, and duplicatereaders were not required for accuracy. The DNA probecontains genetic sequences that are present in both HSV-1and HSV-2 genomic DNAs. Since both virus types are foundin a variety of body sites, it is advantageous that they bedetected by the ISH assay with equal efficiencies. However,this may also be a limitation when typing is necessary forepidemiological surveillance or for a specific diagnosis in apatient. An additional advantage of this particular hybridiza-tion assay is the use of a nonradiolabeled probe to detectHSV target DNA, which avoids the problems associateswith the disposal of radioactive material. It also significantlyimproves the stability of the probe-related reagent andprolongs its shelf-life.

In our hands, the rapidity of results, simplicity of testperformance, and ease of detection of positive results pro-vide an attractive alternative to conventional tissue culturetechnology for the detection and identification of HSV inclinical material.

ACKNOWLEDGMENTS

We thank Mark Kielkucki for help with manuscript preparation,Maryanne Gsell for technical assistance, and William Merz forcritical review.

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