Detection of herpes simplex virus in clinical specimens by DNA hybridization

DIAGNMICROBIOLINFECTDIS 1983;1:117-128

117

Detection of Herpes Simplex Virus in Clinical Specimens by DNA Hybridization David C. Redfield, Douglas D. Richman, Sara Albanil, Michael N. Oxman, and Geoffrey M. Wahl

An assay to detect herpes simplex virus (HSV) DNA in clinical specimens has been developed. It utilizes nucleic acid hybridization with a 32p-labeled DNA probe prepared from a fragment of HSV DNA cloned in a plasmid vector. This assay can detect 5 × 104 plaque-forming units of cell-free HSV and as few as four virus-infected cells. The assay has a sensitivity of 78% and a specificity of 100% compared to virus culture for the detection of HSV in swab specimens from genital lesions. No hybridization is observed with uninfected, varicellu-zoster virus infected, or cytomegalovirus infected cells, and specimens from herpes zoster lesions are uniformly negative. While hybridization with a 32p-labeled probe is not optimally suited for routine diagnostic use, this report establishes the feasibility of using nucleic acid hybridization to detect HSV in clinical specimens.

INTRODUCTION Herpes genitalis has become an increasingly important sexually transmitted disease (CDC report, 1982). It is estimated that 5,000,000 (i.e., 1 in 20) adults in the United States were infected as of 1980 (NIH publ 80-2005) and that up to 250,000 are becoming infected annually (Nahmias and Josey, 1982). Initial genital infection with herpes simplex virus (HSV) results in persistance of virus in the sacral ganglia (Baringer, 1974) and is often followed by recurrent genital infections, with the potential for transmission to sexual partners and newborns. Herpes genitalis has also been linked epidemiologically to cervical cancer (Rawls et al, 1977). Precise and rapid diagnosis of genital HSV infection is desirable to permit the appropriate use of newly available chemotherapeutic agents, the proper counseling of patients with this often vexing clinical problem, and the prudent management of pregnant women. While the clinical features of the disease are well known to most physicians, atypical presentations (Yen et al., 1965; Willcox, 1968; Stone et al., 1977), coexistent infections (Beilby et al., 1968; Fiumara et al., 1980; Sumaya et al., 1980), and masquarading syndromes (Hutfield, 1970) may lead to confusion with respect to the diagnosis. Virus culture is the standard diagnostic test, but it is expensive, time consuming, and unavailable in many diagnostic laboratories. Cytologic or histopathologic diagnosis is helpful in some cases, but the sensitivity of such exami-

From the Departments of Medicine and Pathology, San Diego Veterans Administration Medical Center and School of Medicine, University of California, San Diego, California (D.C.R.; D.D.R.; S.A.; M.N.O.) and the Tumor Virology Laboratory, The Salk Institute, La Jolla, California (G.M.W.). Address reprint requests to: D a v i d C. Redfield, M.D., Infectious Disease Section--111F, VA Medical Center, 3350 La Jolla Village Drive, San Diego, CA 92161. Received December 27, 1982; accepted January 28, 1983.

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nations is generally 50% or less (Morse et el., 1974; Vontver et al., 1979; Brown et al., 1979). Immunofluorescent or immunoperoxidase staining can improve sensitivity considerably, but well-characterized reagents have not been generally available, many specimens are unsatisfactory for examination, and interpretation of results is subjective and requires considerable expertise (Nahmias et al., 1971; Benjamin, 1977). Serologic tests can determine whether or not infection has occurred in the past, but can diagnose acute infection only retrospectively. Moreover, the serologic tests now readily available do not even distinguish prior nongenital HSV infections from genital herpes. Assays for the presence of virus antigens in clinical specimens are currently under development in several laboratories (Cleveland et al., 1982b; Vestergaard and Jensen, 1981) and this approach offers promise for rapid diagnosis. Assays for the presence of virus nucleic acid in clinical specimens also appear promising. Flores et el. (1982) have reported the detection of rotavirus RNA in stool samples by RNA hybridization. Chou and Merigan (1982) have utilized DNA hybridization to detect cytomegalovirus DNA in urine specimens. We describe here the feasibility of using DNA hybridization to detect HSV DNA sequences in clinical specimens using a a2p-labeled DNA probe prepared from a fragment of HSV DNA cloned in a plasmid vector. MATERIALS AND METHODS Cells and Viruses A strain of diploid human flbroblasts (350Q) initiated from newborn foreskin at the Virus Research Unit, Childrens Hospital Medical Center, Boston, MA, was used between passages 18 and 30. Cells were grown in an atmosphere of 5% CO2 in air in Dulbecco's modified Eagle's medium containing 100 U/ml penicillin G and 100 ~g/ml streptomycin (DMEM) plus 10% fetal calf serum (FCS). HSV-1 (F strain) was provided by Dr. Bernard Roizman, University of Chicago, Chicago, IL. HSV-1 (Sanchez strain) from the brain of a patient with encephalitis, and HSV-2 (Wolfe strain) from a genital lesion, were isolated in this laboratory and typed by immunofiltration assay (Cleveland, 1982a). The AD-169 strain of cytomegalovirus (CMV) and the CP5262 strain of varicella-zoster virus (VZV) from the Centers for Disease Control, Atlanta, GA, were used as prototype CMV and VZV. Virus-infected cells were prepared as large pools of monodisperse HSV-1, HSV-2, CMV, and VZV infected 350Q cells (Cleveland et al., 1982a). Cell-free HSV-1 and HSV-2 were prepared from infected cultures of 350Q, as previously described (Redfleld et al., 1981).

Clinical Specimens Specimens were obtained as reported from patients participating in an evaluation of oral acyclovir for treatment of initial or recurrent genital herpes (Reichman et al., 1982). Cotton swab specimens were carried to the laboratory on ice in transport medium consisting of Hanks balanced salt solution containing 1% FCS, 100 U/ml penicillin, 100 ~Lg/ml streptomycin, and 5 ~g/ml amphotericin B; 1 ml for male patients, 2 ml for female patients. Virus isolation was attempted within a few hours of specimen collection and the samples were then stored at - 70°C, except for some specimens from the female patients which were first stored briefly at -20°C. Virus isolates were typed by enzyme immunofiltration assay using type-specific monoclonal antibodies (Richman et al., 1982). On the day that DNA was prepared for hybridization, the specimens were thawed and an aliquot removed for titration of infectivity, as previously described (Cleveland at al., 1982b). Similarly obtained swab

Detection of Herpes Simplex by DNA Hybridization

119

specimens from patients with virus culture-positive herpes zoster were included as controls in some experiments.

Preparation of DNA Blots Samples were prepared according to the method of Tlsty et al. (1982), with minor modifications as specified. Virus-infected Cells Virus-infected and uninfected cells were washed once and resuspended in TD buffer (25 mM Tris [pH 7.4], 140 mM sodium chloride, 5 mM potassium chloride, 0.7 mM dibasic sodium phosphate). Sodium hydroxide was added to 0.3 M and the suspension was incubated for 1 hr at 60°C in polypropylene tubes to degrade the RNA and denature the DNA. An equal volume of 2M ammonium acetate was added and duplicate 0.25 ml aliquots were applied by filtration to 0.8 × 7.0 m m rectangular areas of 0.2 p.m nitrocellulose paper (BA 83, Schleicher and Schuell, Keene, NH) with the aid of a specially designed suction apparatus (Slot Blotter, Schleicher and Schuell, Keene, NH). This "slot blot" configuration concentrates the sample in a small area and facilitates subsequent densitometer scanning.

Cell-free Virus Suspensions of virus in DMEM plus 2% FCS were serially diluted in TD buffer, with sodium hydroxide added to 0.3 M, and were incubated for 1 hr at 60°C. An equal volume of 2M ammonium acetate was added and aliquots were applied to 0.2 ~Lm nitocellulose paper, as described for virus-infected cells.

Clinical Specimens An aliquot of the transport medium containing the specimen was centrifuged for 10 min at 10,000 × g. The supernatant was removed and replaced with an equal volume of TD buffer. This step was necessary in order to reduce the viscosity of some specimens and allow efficient filtration through the nitrocellulose paper. The pellet was resuspended by vortexing, sodium hydroxide was added to 0.3 M, and the sample was incubated for 1 hr at 60°C. An equal volume of 2M ammonium acetate was added and aliquots were applied to 0.2 ~m nitrocellulose paper as described for virusinfected cells. In some cases the specimen supernatant, as well as an uncentrifuged aliquot of the original specimen, were similarly treated and applied to nitrocellulose paper. The nitrocellulose blots were air dried and then baked for 2 hrs at 80°C before hybridization. Plasmid DNA Plasmid pRB131, consisting of the Bam HI restriction endonuclease A fragment (Locker and Frenkel, 1979) of HSV-1 (F strain) inserted into plasmid pBR322 (Post et al., 1980), was generously provided by Dr. Bernard Roizman, University of Chicago, Chicago., IL, in EscherichiQ coli strain C600SF8. The BAM HI A fragment of HSV-1 consists of DNA from map position 0.13 to 0.22 and has a molecular weight of approximately 7 × 106. Isolation of pRB131 DNA from strain C600SF8 by the method of Clewell and Helinski (1969) resulted in a low yield, and thus the plasmid was

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transformed into calcium shocked (Dagert and Ehrlich, 1979) E. Coli strain DH1 (kindly provided by Dr. D. Hanahan, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Significantly better plasmid yields were obtained from strain DH1 using the same method (Clewell and Helinski, 1969). The structure of pRB131 was confirmed by analysis of Barn HI digested plasmid DNA on agarose gels (McDonnell et al., 1977). Plasmid MUA-3, a derivative of pBR322 (Meyerowitz et al., 1980), was similarly prepared from E. coil strain DH1 and used as control plasmid DNA.

Preparation of Probe Radiolabeled probe was prepared by nick translation of plasmid DNA (Rigby et al., 1977). 300 ng of plasmid DNA and 100 p.Ci of 32p-dCTP (specific activity 3000 Ci/m mol New England Nuclear, Boston, MA) in a 20 p,1 reaction mixture yielded probes with a specific activity of 1> 2 × 108 cpm/p.g.

Hybridization Reactions The nitrocellulose blots were prepared for hybridization by incubation in a solution containing 50% formamide, 5X Denhardt's solution, 0.01% SDS, 5X SSPE (0.9 M sodium chloride, 50 mM sodium phosphate [pH 7.7], 5 mM EDTA), and 0.2 mg/ml denatured herring sperm DNA for at least 2 hrs at 42°C. Blot hybridization was carried out in 50% formamide, 10% dextran sulfate, 5X Denhardt's solution, 0.01% SDS, 5X SSPE, 0.2 mg/ml denatured herring sperm DNA, and denatured probe at 107 cprrgml for 18-20 hrs at 42°C (Wahl et al., 1979). Following hybridization, blots were washed first in a solution of 1X SSPE, 0.1% SDS for 10 min at room temperature, next in 0.iX SSPE, 0.1% SDS for 30 min at either 42°C or 60°C, and finally in 0.1X SSPE, 0.1% SDS for 10 min at room temperature (Wahl et al., 1979). Washed blots were exposed overnight at - 70°C to XAR x-ray film (Eastman Kodak Co., Rochester, NY) with a Cronex Hi-Plus intensifying screen (Dupont, Wilmington DE). The autoradiographs were analyzed using a Quick Scan R & D densitometer coupled to a Quick Quant III computer (Helena Laboratories, Beaumont, TX). Signal quantitation was achieved by densitometer analysis of the autoradiographs and computation of an integrated intensity value derived from the area under the curve of the densitometer tracing. In some instances, the DNA sample slots were cut out of hybridized blots and counted in a scintillation counter in vials containing 2 ml Cytoscint (West Chem Products, San Diego, CA).

RESULTS Detection of HSV-1 and HSV-2 Infected Cells by DNA Hybridization The HSV probe (from pRB131) and control probe (from MUA-3) were hybridized to blots prepared with DNA extracted from serial dilutions of virus-infected and uninfected cells (Figure 1). The HSV probe clearly detected viral DNA from as few as 4 HSV-1 or 8 HSV-2 infected cells. The control probe did not hybridize to infected cell DNA, and neither probe hybridized with uninfected cell DNA. The amount of radiolabel bound after hybridization could also be measured by excising the slot from the blot and counting in a scintillation counter; however, autoradiography was more sensitive, owing to the accumulation of radiation during overnight film exposure (data not shown).

Detection of H e r p e s Simplex b y D N A Hybridization

HSV-1 INFECTED CELLS

121

HSV-2 INFECTED CELLS

HSV Control Probe Tntensity Probe

Infected Cel Is

HSV Control Probe Intensity Probe

I000

(304,8)

Number

of

(6043)

500

(2611)

(4305)

250

(2087)

(25o4)

125

(904)

(14761

62

(444)

(547)

31

(171)

.5o)

16

(68)

8

(34)

(791

..............

(67)

4 2 0

FIGURE 1. Autoradiographs of the hybridization of the 32p-labeled HSV and control probes to the DNA from HSV-1 and HSV-2 infected cells. DNA was extracted from mixtures of HSV infected and uninfected 350Q cells, which contained from 1000 to 0 infected cells per 1000 total cells, and applied to nitrocellulose paper as described in Methods. Replicate nitrocellulose blots were hybridized w i t h HSV and control probes, washed at 42°C, and autoradiographs were prepared as described in Methods. Numbers in parentheses represent the integrated intensity values obtained by densitometer scanning. Where no number appears, the intensity value was <10. No intensity values appear for the hybridization reactions between the control probe and DNA from HSV-1 or HSV-2 infected cells because all of these values were <10. Detection of Cell-Free Virus DNA DNA blots were p r e p a r e d from two strains of cell-free HSV-1 and one strain of HSV2. The HSV probe h y b r i d i z e d w i t h all three virus DNAs, permitting the detection of as little as 5 × 10 4 PFU of the HSV-1 strains and 3 x 10 5 PFU of the HSV-2 strain (Figure 2). No h y b r i d i z a t i o n was observed w i t h the control probe.

FIGURE 2. Autoradiographs of the hybridization of the 32p-labeled HSV and control probes to the DNA from cell-free HSV-1 and HSV-2. DNA was extracted from the indicated number of plaque-forming units (PFU) of virus and applied to nitrocellulose paper as described in Methods. Replicate nitrocellulose blots were hybridized w i t h HSV and control probes, washed at 42°C, and antoradiographs prepared as described in Methods. Numbers in parentheses represent the integrated intensity values obtained by densitometer scanning. Where no number appears, the

intensity value was <10. No intensity values appear for the hybridization reactions between the control probe and DNA from HSV-1 or HSV-2 because all of these values were <10. r---~

HSV- 1 (F)

PFU

HSV Control Probe Intensity Probe

5 x 106 5 x 105 5 x 104 5 xlO 3

(8335) (2966) (872) (40)

i

i

HSV-I (Sonchez) PFU

5 x 106 5 x IO5 5 x 104 5 xlO 3

HSV Control Probe Intensity Probe

i

HSV-2 (Wolfe) PFU

HSV Control Probe Intensity Probe

(7625)

(1(51o) 14)

3 x IO 5

3 x 104 3 x I03

(105)

I

122

D . C . R e d f i e l d e t al.

Detection

of HSV DNA in Clinical

Specimens

Having determined the sensitivity of the hybridization assay for virus-infected cells a n d c e l l - f r e e v i r u s , w e a t t e m p t e d to d e t e c t H S V D N A i n c l i n i c a l s p e c i m e n s . T h e r e s u l t s o b t a i n e d w i t h 4 0 c u l t u r e - p o s i t i v e s p e c i m e n s ( d e s c r i b e d i n T a b l e 1) a n d 3 0 c u l t u r e - n e g a t i v e s p e c i m e n s a r e s h o w n i n t h e a u t o r a d i o g r a p h s i n F i g u r e s 3 a n d 4, respectively. A positive result was recorded when hybridization with HSV probe

T A B L E 1. V i r u s C u l t u r e - p o s i t i v e

Clinical Specimens V i r u s infectivity

Specimen number

Source °

Type b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2O 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

I I I I I I II I I I I I I I I I I I I IIl III III III III III III III III III Ill Ill Ill Ill III III III Ill III Ill IV

2 2 2 2 2 2 2 2 2 2 2 2 2 nd nd 2 nd nd nd 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1

Titer c 2.0 2.5 <2.0 4.5 5.0 I>5.5 5.0 I>5.5 <2.0 2.0 <2.0 I>5.5 3.5 <2.0 <2.0 2.5 <2.0 <2.0 <2.0 2.0 2.0 />5.5 4.5 <2.0 I>5.5 <2.0 />5.5 4.5 I>5.5 4.5 4.5 4.5 4.5 5.0 3.5 3.5 ~>5.5 ~>5.5 3.5 <2.0

~I = female genital lesion; II = cervix; III = male genital lesion; IV = male urethra ~1 = HSV-1; 2 = HSV-2; n d = not done qog,o TCIDso per ml

Control Spec. HSV no. Probe Intensity Probe

3

20 21 22

5981 ]

4

23

:1513)

5

24

6

25

(4t) 2263)

7

26

(2tl)

8

27

2348)

1

(70)

2

I588)

9

28

2437)

10

29

5427)

11

50

1586)

t2

51

:1251)

13

32

I510)

14

33

[143)

15

34

(1517)

'16

35

(347)

17

36

(1456)

19

I c2533)

19

~ii~i~!i/~i~!~!~;

123

Spec~ HSV Control no. Probe Intensity Probe

~i~i~i~i!~i~~i~!~i ~

37

(1607)

58

(2745)

59

(969)

40

FIGURE 3. Autoradiographs of the hybridization of the 3zP-labeled HSV and control probes to the DNA from culture-positive clinical specimens. DNA was extracted and applied to nitrocellulose paper as described in Methods [from 0.3 ml aliquots of specimens from female patients, numbers 1-19, and from 0.2 ml aliquots of specimens from male patients, numbers 20-40). Replicate nitrocellulose blots were hybridized with HSV and control probes, washed at 6O°C, and autoradiographs prepared as described in Methods. Numbers in parentheses represent the integrated intensity values obtained by densitometer scanning. Where no number appears, the intensity value was <10. No intensity values appear for the hybridization reactions between the control probe and DNA from these specimens because all of these values were <10. FIGURE 4. Autoradiographs of the hybridization of the 32p-labeled HSV and control probes to the DNA from culture negative clinical specimens. Specimen numbers 41-50 are cervical swab specimens, 51-60 are female genital lesion swab specimens, 61-65 are male urethral swab specimens, and 66-70 are male genital lesion swab specimens. DNA was extracted and applied to nitrocellulose paper as described in Methods. Replicate nitrocellulose blots were hybridized with HSV and control probes, washed at 60°C, and autoradiographs prepared as described in Methods. Numbers in parentheses represent the integrated intensity values obtained by densitometer scanning. Where no number appears, the intensity value was <10. (It should be noted that for specimen 46 a trace signal was visible with the control probe, but gave an intensity value of <10.) FemaleCervicalSpecimens----~

[

Spec. HSV

Control

no. Probe Intensity Probe Intensity

41 42 43 44 45 46

47 48 49 50

I

i--Male Specimens~

Control no. Probe Intensity Probe Intensity 51

Spec, HSV Control no. Probe Probe

I

FemoleGenital Specimens

Spec, HSV

52 (50)

(55)

(36)

53 54 55

(281 (29) (35)

(32)

56

(26)

57 58

(2e}

(115)

(89)

59

(46)

(55)

(22)

60

(34)

.......

(28)

7O

124

D.C. Redfield et al.

produced a strong signal (intensity value > 40), while hybridization of the duplicate DNA slot with the control probe produced either no signal or a trace signal (intensity value < 10). 11 of 19 culture-positive specimens from female patients and 19 of 21 from male patients were scored as positive (Figure 3). Of the 10 remaining culturepositive specimens, one of the male patients' specimens (number 21), and several of the female patients' specimens (numbers 3, 10, 14, 15, and 17), produced a weak signal with the HSV probe and no signal with the control probe. A repeat assay using a larger sample from some specimens which were initially negative resulted in the detection of HSV DNA in one additional specimen, number 3 (data not shown). Hybridization thus identified HSV DNA in 31 of 40 (78%) virus culture-positive specimens. When these results were correlated with the titer of infectious virus in each specimen, it was observed that HSV DNA was detected in all samples with a titer of 9102.5 TCIDso/ml, 90% of those with a titer of/>102'° TCIDso, but in only 40% of those with a titer of <102° TCIDso/ml (Figure 5). Hybridization to blots of DNA from culture-negative specimens yielded one specimen (number 49) with a moderate signal with both probes and several others with weak signals with one or the other

FIGURE 5. Correlation of specimen infectivity titer with the presence of HSV DNA detectable by hybridization with the HSV probe. The net HSV specific intensity value is the difference between the intensity value obtained by hybridizing DNA extracted from an aliquot of the specimen with the HSV probe and the intensity value obtained by hybridizing that DNA with the control probe. The dashed line at the net HSV specific intensity value of 40 represents the arbitrary cut off value differentiating hybridization positive and negative specimens, e-male genital lesion swab specimen; A-male urethral swab specimen; o-female genital lesion swab specimen; A-cervical swab specimen. 10,000 8

1000 o

100 L~J I'--

o

,v

i

°

10 o

Z

o

eoo@ AeA OAO AA I

.(i

I

I

Negative Positive 1020 (30 Specimens) but < 102.0

I

I

103.0

I

I

104.0

I

I

t¢

105.0"" >105.5

INFECTIVITY TITER (TCIDso/ml}

Detection of Herpes Simplex by DNA Hybridization

125

probe (Figure 4). However, no culture-negative specimen gave even a trace signal with the HSV probe in the absence of some signal with the control probe, and none was therefore scored as positive. Of note, clinical specimens treated directly with sodium hydroxide were sometimes viscous and thus very slow in filtration. This problem was overcome by centrifuging the specimen and resuspending the pellet in buffer prior to DNA extraction. This procedure did not result in any detectable loss of HSV DNA. In fact it increased sensitivity by making practical the application to nitrocellulose of DNA from a larger aliquot of some specimens. Specificity of the HSV DNA Probe DNA blots were prepared from cells infected with VZV and CMV in order to detect any possible homology between the HSV probe and other human herpesviruses. Blots of DNA prepared from 10 4 VZV and CMV infected cells failed to hybridize with the HSV probe (data not shown). In addition, blots of DNA prepared from VZV culturepositive swab specimens from the lesions of 5 patients with acute herpes zoster failed to hybridize with the HSV probe (data not shown). DISCUSSION Direct detection of HSV DNA in clinical specimens has been achieved by hybridization using a ~2p-labelled DNA probe prepared from a cloned fragment of HSV DNA. This technique had a sensitivity of 78% and a specificity of 100% when compared to virus culture. Semiquantitative results could be obtained by direct visualization of the autoradiographs, and quantitative results could be obtained by densitometer analysis. Weak hybridization with both the HSV and control probes was observed with some of the culture-negative specimens. In none of these, however, was the signal significantly stronger with the HSV probe than with the control probe. It is possible that these weak signals with virus culture-negative specimens are, in part, due to the presence of bacteria containing ~-lactamase sequences, since such sequences are present in the plasmid vectors used for both the HSV and control probes. Alternatively, weak hybridization signals may result from nonspecific interactions with substances present in the swab specimens (Southern, 1979). It is noteworthy that these weak signals with culture-negative samples were obtained exclusively with specimens from female patients, in which higher levels of bacterial contamination, as well as contamination with urogenital secretions, would be expected. The hybridization technique described is not yet as sensitive as virus culture for the detection of HSV in these clinical specimens. Nevertheless, this approach offers several potential advantages. Hybridization can accomplish both virus detection and identification in a single step, whereas some additional procedure for virus identification is required after isolation of an infectious agent in cell culture. In addition, the use of HSV type-specific probes (Stalhandske and Petterson, 1982) should make it possible to simultaneously detect, identify, and type HSV with a single procedure. However, the greatest advantage of hybridization over virus isolation is its potential for achieving rapid viral diagnosis. Virus isolation in cell culture generally takes a minimum of 1-2 days; while the majority of positive specimens induce characteristic cytopathic effects (CPE) by 3 days after inoculation, some require longer periods of incubation. In an attempt to prevent neonatal herpes, it is now recommended that pregnant women with a history of genital herpes or conjugal exposure to genital herpes be monitored with virus cultures in the latter part of pregnancy, so that delivery may be accomplished by Caesarian section if HSV is present in the birth

126

D.C. Redfield et al.

canal at term (Brann and Mortimer, 1980; Kibrick, 1980). However, the interval between culture acquisition and the appearance of viral CPE may reduce the efficacy of this approach, since the presence of HSV may not be recognized until after delivery (Grossman et al., 1981). Furthermore, since a medical history and physical examination appear to identify fewer than half of the pregnancies at risk (Whitley et al., 1980), a major reduction in the occurrence of neonatal herpes may require the screening of all pregnant women. Clearly, a more rapid and economical method is needed to permit evaluation at the time of labor. Rapid diagnosis of genital HSV infection is also more important as we enter an era of effective antiviral drug therapy. Recent studies with acyclovir have shown significant benefit when treatment was begun early in the course of initial infection (Mertz et al., 1982). Rapid detection and quantitation of virus by hybridization might also provide an effective means by which to monitor antiviral therapy. Several improvements are needed before the DNA hybridization technique we describe here can be widely applied to clinical diagnosis. Sensitivity must be improved so that it is at least comparable to that of virus culture, a substitute must be found for the 32p label currently employed, and the time required must be reduced so that results are available within a few hours. With the present HSV probe, pRB 131, the assay is more sensitive for HSV-1 than for HSV-2; only 78% sensitivity was obtained with clinical specimens of HSV-2. (The sensitivity of the assay for HSV-1 in clinical specimens has not yet been determined.) The relative sensitivity of the assay for HSV-2 DNA can be increased by utilizing, as probe, segments of the HSV2 genome. Furthermore, in view of the frequency with which HSV-1 is now isolated from patients with genital herpes {Kalinyak et al., 1977), it will be necessary to utilize a probe or probes which provide maximal sensitivity for both HSV-1 and HSV-2. This might be accomplished by the incorporation of segments of DNA from both virus serotypes. Hybridization with a 32p-labeled DNA has drawbacks. Such probes have a short half-life and thus must be prepared frequently. Moreover, radiation exposure and disposal are additional disadvantages. Nonradioactive probes with relatively long shelf lives are essential. Biotinylated probes (Langer et al., 1981) that function well in hybridization reactions and can be detected by immunofluorescence, immunoperoxidase staining, or affinity binding with avidin conjugates have recently been developed. Such probes have already been used to detect parvovirus, papovavirus, HSV, adenovirus, and retrovirus nucleic acids in situ (Brigati eta]., in press) and this form of labeling can be substituted for 32p in our assay (unpublished results). Finally, for rapid viral diagnosis, the interval from specimen collection to completion of the hybridization assay must be shortened so that results are available on the same day. Preliminary results indicate that the use of a biotinylated probe will enable us to shorten the assay time from its current 36-48 hrs so that results can be obtained within 6 hrs of specimen acquisition. With modifications, detection of viral nucleic acids by hybridization should provide a rapid and sensitive alternative to virus culture for routine diagnosis.

We thank Bernard Roizman, the University of Chicago, Chicago, IL for many helpful discussions and for providing the plasmid cloned HSV sequences. We thank Walter Eckhart, Salk Institute, La Jolla, CA for advice and encouragement. This work was supported by the Veterans Administration, by research contract AI-22681 from the National Institute of Allergy and Infectious Diseases, and by NIH grant I RO1 GM27754 from the Institute of General Medicine. Dr. Redfield is the recipient of National Research Service Award 1-F32-EYO5589-01 from the National Eye Institute.

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