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5. Seidel-Dugan C, Ponce de Leon M, Friedman HM, Eisenberg RJ, Cohen GH. Identification of C3b-binding regions on herpes simplex type 2 glycoprotein C. J Virol 1990;64: 1897-906. 6. Friedman HM, Vee A Digglemann H, et al. Use ofa glucocorticoid-inducible promoter for expression of herpes simplex type I glycoprotein gC I, a cytotoxic protein in mammalian cells. Mol Cell Bioi 1989;9:2303-14. 7. Seidel-Dugan C, Ponce de Leon M, Friedman HM, et al. C3b receptor activity on transfected cells expressing glycoprotein C of herpes simplex virus type I and 2. J Viral 1988;62:4027-36. 8. Smiley ML, Hoxie lA, Friedman HM. Herpes simplex virus type I infection of endothelial, epithelial, and fibroblast cells induces a receptor for C3b. J Immunol 1985; 134:2673-8.
10. Smiley ML, Friedman HM. Binding of complement component C3b to glycoprotein C is modulated by sialic acid on herpes simplex virus type I infected cells. J Virol 1985;55:857-61. II. Devereux JR, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 1984; 12:387-95. 12. Corbi AL, Kishimoto TK, Miller LJ, Springer T A. The human leukocyte adhesion glycoprotein Mac-I (complement receptor type 3, CDllb) ll' subunit. J Bioi Chem 1988;263:12403-1 I. 13. van Strijp JAG, van Kessel KPM, van der Tol ME, Verhoef J. Complement-mediated phagocytosis of herpes simplex virus by granulocytes. J Clin Invest 1989;84: 107-12. 14. Harold BC, WuDunn 0, Soltys N, Spear PG. Glycoprotein C of herpes simplex virus type I plays a principal role in the adsorption of virus to cells and in infectivity. J Virol 1991;65: 1090-8. 15. Langeland N, Oyan AM, Marsden HS, et al. Localization on the herpes simplex virus type I genome of a region encoding proteins involved in adsorption to the cellular receptor. J Virol 1990;64: 1271-7.
A Double-Blind, Placebo-Controlled Cytogenetic Study of Oral Acyclovir in Patients with Recurrent Genital Herpes Donald Clive, Lawrence Corey, Richard C. Reichman, L. Gray Davis, and John C. Hozier
Burroughs Wellcome, Research Triangle Park. North Carolina; Departments of Laboratory Medicine and Medicine, University of Washington, Seattle; Infectious Diseases Unit. University of Rochester School of Medicine, New York; Applied Genetics Laboratory, Melbourne, Florida
The antiherpes drug acyclovir breaks chromosomes in vitro at millimolar concentrations and at highly toxic doses in rodents but does not induce single-gene mutations. Recurrent genital herpes patients were examined to determine if such chromosomal damage occurs in peripheral lymphocytes during acute or chronic acyclovir therapy. Patients were randomly assigned to receive acyclovir suppressively and for recurrences, placebo suppressively and acyclovir for recurrences, or placebo suppressively and for recurrences (n ~ 20 for each group; all treatment doubleblind). Normal volunteers and acyclovir-treated cultures served as additional controls. Cytogenetic analyses were done at enrollment (pretreatment), on day 5 of acute acyclovir or placebo treatment for the first postenrollment recurrence (postacute), and at the end of a year on study (postchronic). Cells in metaphase, 150 for each patient, were examined at each time point for structural and numerical chromosomal abnormalities. No cytogenetic effects of chronic or acute oral acyclovir treatment were found relative to lifestyle controls, pretreatment controls, or placebo treatment.
Acyclovir (ACV; 9-(2-hydroxyethoxymethyl)guanine; molecular weight, 225.2), a potent inhibitor of herpes simplex virus types I and 2 (HSV-I, -2), has extremely low toxicity
Received 4 February 1991; revised 23 May 1991. Presented in part: Environmental Mutagen Society meeting. Charleston. South Carolina. March 1988 (abstract 55). Informed consent was obtained from patients or guardians, and guidelines for human experimentation of the ethical committees of the University of Rochester School of Medicine and University of Washington Department of Laboratory Medicine were followed. Grant support: National Institutes of Health (AI-2038I ). Reprints or correspondence: Dr. Donald Clive. Burroughs Wellcome Co., Research Triangle Park, NC 27709. The Journal of Infectious Diseases 1991;164:753-7 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6404-0018$01.00
for uninfected host cells [I, 2]. This high therapeutic index results from the specific activation of ACV to its monophosphate by a herpes-specified thymidine kinase; in herpes-infected cells, this can subsequently be converted to the di- and triphosphates by normal cellular enzymes [3]. ACV triphosphate acts by preferential inhibition of the herpes-specified DNA polymerase [3]. The genetic toxicology of ACV in vitro and in rodent in vivo studies has been presented elsewhere [4]. ACV does not induce single-gene mutations in a variety of in vitro microbial or mammalian cell systems at concentrations up to and often exceeding 2500 ,ttg/ml but is clastogenic at concentrations > 125 .tLg/ml in vitro or the equivalent plasma levels in vivo. Such high plasma levels crystallize out in renal tubules and lead to extensive obstructive nephropathy [5]. Although
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9. Gaither TA, HammerCH, Frank MM. Studies on the molecularmechanisms ofC3b inactivation and a simplified assay for b IH and the C3b inactivator (C3nINA). J Immunol 1979; 123: 1195-233.
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typically lethal to about one-third of test animals within 2-4 days of single intravenous dose administration, this highdose effect is masked by the typical sacrifice schedule of most in vivo short-term studies. This specificity for inducing chromosomal damage formed the basis for investigating the genotoxicity of ACV in humans in a cytogenetic study. The present investigation was part of a double-blind, placebocontrolled study of the ability of oral acyclovir to suppress the frequency of recurrent genital herpes outbreaks [6].
Patients and Methods
To ensure blind scoring, this blood specimen was assigned a number previously deleted from the clinical sequence and was timed to "arrive" at the cytogenetics laboratory at the appropriate time. Experimental design method for the control ofbias. Enrolled patients were assigned a sequential patient number that randomly and blindly placed them into one of the principal treatment groups. Patients were treated with ACV or placebo suppressively and during each recurrence according to their group assignment. In addition, non-herpes infected volunteers were selected at each clinical center and assigned to group IV. This last group received no drug and served as matched lifestyle controls for the incidence of chromosome damage. At least 20 patients were enrolled in each group. Each blood sample was assigned a unique and sequential specimen number. This number was the only information accompanying the blood sample when cytogenetic analyses were done. Codes were broken at Burroughs Wellcome Genetic Toxicology Laboratory only after the data had been transmitted and recorded. Patients were screened for occupation, recent viral diseases, use of social drugs, medications, smoking, and other factors likely to influence the outcome of the study (table 1). Cytogenetics. Each blood specimen was cultured in quadruplicate and prepared for cytogenetic analysis in duplicate. The medium used was RPMI 1640 supplemented with 20% fetal calf serum, 100 units/rnl penicillin, 100 Ilg/ml streptomycin, and 2 mM L-glutamine. The following were added to 4.7-ml aliquots of this freshly supplemented medium in each of four T-25 flasks: 0.3 ml of heparinized blood, one drop of heparin (at 1000 units/ ml), and 200 III of freshly reconstituted phytohemagglutinin (M-Form; GIBCa, Grand Island, NY). Cultures were incubated at 37°C for 42-48 h. Three hours before the end of the culture (groups I-IV) or treatment (group V) period, the quadruplicate cultures were reTable 1.
Demographics for patients treated orally with acyclovir. Group
Age Mean ± SD Range Sex Male Female Race White Other Smoking Yes No Not ascertained Oral contraceptives (females only) Use Don't use
II
III
IV
32 ± 7 22-50
34 ± 10 22-62
32 ± 6 23-43
31 ± 5 23-42
17 (63) 10 (37)
18 (62) II (38)
15(62) 9 (38)
12 (48) 13(52)
25 (93) 2 (7)
28 (97) I (3)
23 (96) I (4)
24 (96) I (4)
II (41) IS (56) I (4)
9 (31) II (38) 9 (31)
5 (21) 12 (50) 7 (29)
9 (36) 15 (60) I (4)
5 (50) 5 (50)
3 (27) 8 (73)
3 (33) 6 (67)
I (8) 12 (92)
NOTE. Groups: I, acyclovir suppressively andfor recurren~es; II, placebo suppressively and acyclovir for recurrences; III, placebo ~uppresslvely andfor recurrences; IV, lifestyle controls. Data are no, (%) unless indicated.
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Test substances. ACV capsules (200 mg; Burroughs Wellcome) were used to treat enrolled patients. ACV was also used to dose blood cultures set up for positive controls. For placebo control, lactose in capsule formulation identical with that of ACV was used. Subjects. Subjects were those enrolled in a placebo-controlled study (protocol 137) in 1984-1987 that compared suppressive and acute oral ACV therapy in the management of recurrent genital herpes [6]. This study was done at two centers: the University of Washington and the University of Rochester School of Medicine. Patients diagnosed with culture-documented recurrent genital herpes were enrolled in this study at a time when they were asymptomatic. On enrollment they were randomized into one of three treatment groups: group I, 400 mg of ACV twice daily suppressively (i.e., throughout the l-year duration of the study or until patients dropped out) and 200 mg of ACV five times daily acutely (i.e., for 5 days during each outbreak) for recurrences; group II, placebo suppressively and ACV for recurrences; and group III, placebo suppressively and for recurrences. In addition, non-herpes infected volunteers were selected at each clinical center to serve as lifestyle controls (group IV). The lifestyle controls were confirmed as antibody-negative to HSV-I and -2. These volunteers were of similar age to groups I-III and were selected from personnel within the clinical laboratories at the two medical centers. They received no treatment and were on no other therapy. An in vitro positive control group (group V) was produced at the cytogenetic laboratory (see below). Blood specimens. The first of three blood specimens for cytogenetic analysis was drawn at enrollment and served to determine each patient's pretreatment (i.e., control) level of cytogenetic damage. A second specimen was collected on day 5 of acute ACV or placebo treatment for the first postenrollment recurrence and served to measure cytogenetic effects of this acute ACV treatment. The third and final specimen was collected at the end ofa year on study and served to measure cumulative cytogenetic effects ofa chronic suppressive therapy. Each specimen consisted of 10 ml of blood drawn into a Venoject or Vacutainer tube containing sodium heparin. Each blood specimen was shipped overnight from the two clinical laboratories to the laboratory performing the cytogenetic analysis (Applied Genetics Laboratories). For group V positive controls, blood was drawn at predetermined intervals from a volunteer at Applied Genetics Laboratories, cultured, and treated with ACV ( 175 Ilg/ ml for 24 h) by a person not involved in the cytogenetic analysis.
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Results Table I summarizes the demographic characteristics of each treatment and control group. No significant differences were detected for age, sex, race, smoking, or incidence of recent viral illnesses. Somewhat fewer women used oral contraceptives in the lifestyle control group than in the three treatment groups. Although there was significant nonascer-
tainment for some parameters, this is not believed to have led to any bias that might have affected the outcome of this study. Table 2 summarizes, for each group of patients or controls, the cytogenetic aberration results at the time of enrollment, after treatment for the first recurrence on study, and after I year of therapy. No statistically significant differences in number of chromosomal aberrations over time or between placebo and any of the ACY recipients were noted. Similarly, the frequencies ofchromosomal aberrations were equal in the lifestyle controls and all treatment groups. No patient exhibited >6.3 chromosome abnormalitiesflOO cells. No statistically significant differences in mitotic indices were noted over time, between placebo and any group of ACV recipients, or between virus-infected and uninfected controls. Table 3 summarizes the incidences of numerical chromosome damage (hypodiploidy, hyperdiploidy, and polyploidy) by treatment group and time. There were no significant differences among groups except for an increase in hyperdiploidy and a possible reduction in hypodiploidy in group V (positive controls). The latter may reflect the fresher, untraveled blood specimens used in this group. Group V consisted of seven positive control specimens treated in vitro with ACY. The mean (±SD) percentage of aberrant cells in this group was 6.7 ± 2.9; the mean (±SD) number of aberrations per 100 cells was 8.4 ± 3.9. This reflects the expected in vitro effects of ACY as described previously [4]. Overall, the incidence of structural and numerical chromosome alterations was not significantly affected by chronic or acute ACY treatment relative to either enrollment values,
Table 2. Lack of chromosome breakage after acute and chronic acyclovir treatment. % aberrant cells Group, time I, Pretreatment
Postacute Postchronic II, Pretreatment Postacute Postchronic III. Pretreatment Postacute Postchronic IV, Lifestyle controls V, Positive controls
n
27 19 24 28 27 17 24 22 15 25 7
Mean ± SO 0.8 0.8 0.6 1.2 1.2 0.5 0.8 1.0 0.8 0.8 6.7
± ± ± ± ± ± ± ± ± ± ±
0.8 1.1 0.6 1.3 l.l 0.5 1.0 1.2 0.6 1.0 2.9
No. aberrations/ 100 cells Range
Mean ± SO
Range
0.0-2.7 0.0-4.7 0.0-2.0 0.0-5.7 0.0-4.2 0.0-1.3 0.0-3.3 0.0-4.7 0.0-2.0 0.0-3.3 3.3-10.7
0.9 ± 0.8 0.9± 1.1 0.6 ± 0.6 1.2 ± 1.3 1.4 ± 1.6 0.5 ± 0.5 1.0 ± 1.3 1.3 ± 1.8 0.8 ± 0.6 0.9±1.1 8.4 ± 3.9
0.0-3.3 0.0-4.7 0.0-2.0 0.0-5.7 0.0-6.3 0.0-1.3 0.0-4.0 0.0-6.3 0.0-2.0 0.0-3.3 4.0-13.3
Mitotic indices, Mean ± SO 1.6 ± 1.2 1.8 ± 1.5 1.5 ± 1.0 1.5±1.1 1.3 ± 1.0 1.4 ± 0.8 1.6 ± 1.8 1.3±1.1 1.5 ± 0.9 2.1±1.9
NOTE. Groups: I, acyclovir (400 mg orally twice daily for I year) suppressivelyand acyclovir (200 mg orally five times daily for 5 days) for recurrences; 11. placebo suppressivelyand acyclovir for recurrences; III, placebo suppressively and for recurrences; IV. laboratory personnel from the two centers; V. blood from normal volunteers treated in vitro with acyclovir (175 ltg/mi. 24 h). Pretreatment blood was sampled at enrollment: postacute, day 5 of treatment for first postenrollment recurrence; postchronic, at end of I year on study. Actual number of days on study (±SD): group 1,365 ± 82; group II. 327 ± 64; group III, 325 ± 88. Lower number of patients after first recurrence is due to dropouts and to absence of recurrences in some patients; lower number at end of study is due to dropouts.
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duced to duplicates by combining in pairs; these duplicate preparations were processed separately thereafter. Demecolcine was added to each flask to a final concentration of 0.2 J,Lg/ml and incubation continued. After 3 h of mitotic arrest, the cells were harvested using 0.075 M KCI and 3: I methanol-glacial acetic acid fixative, dropped onto clean slides, and air-dried overnight. Slides were stained with 2% Giemsa stain in phosphate buffer. One hundred fifty cells in metaphase were examined for each culture. Structural and numerical chromosomal abnormalities and microscope stage vernier coordinates for each metaphase were recorded. The mitotic index (percentage of cells undergoing mitosis, based on 1000 cells counted) was also recorded for each culture. For classification of cytogenetic aberrations, the following structural and numerical chromosomal abnormalities were characterized: chromosome gains and losses, chromatid-type aberrations, chromosome-type aberrations, and severely damaged cells (;;::'10 scorable aberrations). Chromatid and chromosome-type aberrations were further subdivided into deletions and interchanges. By convention, chromatid and chromosome gaps and pulverized chromosomes and cells were recorded but not included in the aberration analysis. Statistical analysis. Kruskal-Wallis tests were used to test for differences between treatment groups and for effects within each group resulting from acute or chronic treatment.
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Table 3.
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Lack of numerical chromosome effects after acute and chronic acyclovir treatment. % Numerically abnormal cells
Group. time
NOTE.
Hypodiploid
27 19 24 28 27 17 24 22 IS 25 7
14.1 ± 6.9 15.6 ± 14.1 10.2±4.5 15.1 ± 8.4 13.3 ± 9.7 10.0 ± 7.3 14.2 ± 7.0 19.4 ± 20.3 10.8 ± 4.8 12.3 ± 6.6 6.9 ± 3.0
Hyperdiploid 0.2 ± 0.5 ± 0.6 ± 0.4 ± 0.4 ± 0.6 ± 0.3 ± 0.6 ± 0.5 ± 0.6 ± 1.6 ±
0.5 0.5 0.9 0.6 0.7 1.1 0.4 0.9 0.6 0.7 0.8
Polyploid 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
± 0.4
± 0.0 ± 0.0 ± 0.0
± 0.0 ± 0.0
± 0.0 ± 0.2 ± 0.0 ± 0.0
± 0.0
See footnote to table 2.
equivalent placebo control group values, or lifestyle control values.
Discussion Analysis of structural and numerical alterations and group mitotic indices showed no effect of chronic or acute oral ACV treatment in recurrent genital herpes patients relative to lifestyle controls, pretreatment controls, or placebo treatment. It is instructive to consider what factors might be involved in this lack of response. The simplest and most obvious explanation for these negative effects is that the mean peak plasma levels of ACV (0.83 and 1.61 JLg/ml after 200- and 600-mg doses, respectively, six times daily) were too low to exert any clastogenicity, particularly in light of the threshold effect seen at ,..." 125 JLg/ml in the in vitro cytogenetic study [4]. However, this clastogenicity of ACV only at high (i.e., millimolar) concentrations in vitro or at comparably high plasma levels in vivo in non-herpes infected systems [4] may not be relevant to the clinical situation, since it presumably proceeds by conversion of ACV to the monophosphate metabolite by reversing the activity of a cellular 5'-nucleotidase. It could be argued that therapeutic levels ofACV in conjunction with the far more efficient (i.e., at micromolar levels) HSV thymidine kinase within an infected cell, followed by cell contact-mediated transport of the phosphorylated ACV from these cells to target peripheral lymphocytes, might cause an increase in chromosomal damage in the latter. However, this possibility receives no support from the present study (tables 2 and 3). Other aspects of the clinical situation might be anticipated to induce clastogenic effects. For instance, an increase in cytogenetic damage might be anticipated as a result of the
viral infection itself, for a number of reasons. Viruses per se are clastogenic [7]; also, untreated patients infected with HSY-l or -2 have been reported to have elevated levels of sister-chromatid exchanges (SCEs) [8], and SCEs are often correlated with the more traditional and genetically better understood chromosomal aberrations measured in this study. In addition, certain types of behavioral stress are genotoxic in rodents [9-11], and these recurrent genital herpes patients may be stressed. It is possible that the above-mentioned increase in SCEs seen in HSV-infected patients resulted from this associated stress rather than from a direct effect of the virus on the genome. No indication of cytogenetic damage was seen in the pretreatment samples compared with those of the lifestyle controls (1. 1 ± 1.2 aberrations/ 100 cells for pooled recurrent genital herpes patients from groups I-III [n = 79] vs. 0.8 ± 1.1 aberrations/ 100 cells for HSV-free lifestyle controls [n = 25]). Thus, for these patients, neither the viral infection nor the stress that might be associated with it were capable of inflicting measurable cytogenetic damage in peripheral lymphocytes. Finally, if ACV (or simply being enrolled in this study) suppresses recurrences, their associated stress, and hence their clastogenicity, it might be argued that this reduction could be masking an equal induction of chromosome breakage by ACV. This is unlikely for two reasons: first, chromosome damage in peripheral lymphocytes persists much longer than the duration of this study, so that many of the virus- (and possibly stress-) induced cytogenetic lesions would not disappear, and any ACY-induced damage would be additive; second, as mentioned above, there was no evidence that such a virus- or stress-related chromosomal effect existed in these patients at entry. In conclusion, this study failed to demonstrate (under conditions that detected an in vitro effect) any significant effect of either acute or chronic oral therapy with ACV on the inci-
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I. Pretreatment Postacute Postchronic II. Pretreatment Postacute Postchronic III, Pretreatment Postacute Postchronic IV, Lifestyle controls V, Positive controls
n
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dence of structural or numerical chromosome damage in peripheral lymphocytes of recurrent genital herpes patients.
6. Mattison HR, Reichman RC, Benedetti J, et al. Double-blind, placebocontrolled trial comparing long-term suppressive with short-term oral acyclovir therapy for management of recurrent genital herpes. Am J Med 1988;85:20-5.
References
7. Gershenson SM. Viruses as environmental mutagenic factors. Mutation Res 1986;167:203-13. 8. Ghosh R, Ghosh PK. Sister-chromatid exchanges in herpes simplex infection. Mutation Res 1983; 119:303-8. 9. Seredenin SB, Durnev AD, Vedernikov AA. Effect of emotional stress on the frequency ofchromosomal aberrations in mouse bone marrow cells. Bull Exp Bioi Med 1980;89:972-3. 10. Fischman HK, Kelly DD, and Rainer JD. Behavioral stress and sister chromatid exchanges [abstract]. Environ Mol Mutagen 1985; 7( suppl 3): 51. II. Kelly DD, Pero R W, Fischman HK. Stress-induced chromosomal alterations: sister chromatid exchanges and unscheduled DNA synthesis [abstract 198.11]. In: Abstracts, 15th annual meeting, Society for Neuroscience (Dallas). Washington, DC: Society for Neuroscience, 1985; I 1:664.
Extended Duration of Herpes Simplex Virus DNA in Genital Lesions Detected by the Polymerase Chain Reaction Richard W. Cone, Ann C. Hobson, Janet Palmer, Michael Remington, and Lawrence Corey
Departments of Laboratory Medicine and Medicine, University of Washington, Seattle
To evaluate the utility of the polymerase chain reaction (PCR) for documenting herpes simplex virus (HSV) in persons with reactivated genital lesions viral isolation was compared with a recently developed PCR method. Three women experiencing four episodes of recurrent genital herpes were followed for 10 days per episode with daily examination and duplicate swabs of the lesions, one for HSV culture and one for PCR. HSV type 2 was cultured from three of four episodes and the mean duration of viral isolation from recurrent genital lesions was 2.6 days. PCR detected HSV DNA from lesion swabs during all four episodes, and HSV DNA was positive for an average of 6.8 days. HSV DNA was demonstrated in ulcerative lesions on 15 of 17 days versus 3 of 17 days by viral isolation (P < .01). HSV PCR became negative when the lesions reepithelialized. These data suggest that PCR is a more sensitive measure of HSV infection than routine viral culture and that PCR detects the presence ofHSV at times when culture is negative.
Herpes simplex virus (HSV) infection is the most common cause of genital ulcerations among patients visiting sexually
Received 12 March 1991; revised 28 May 1991. Presented in part: 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, October 1990 (abstract 199). Informed consent was obtained from the patients studied, and human experimentation guidelines of the University of Washington were followed in the conduct of this research. Grant support: National Institutes of Health (AI-20381). Reprints or correspondence: Dr. Richard Cone, Children's Hospital and Medical Center. 4800 Sand Point Way N.E., Room D-536, Seattle, WA 98105.
The Journal of Infectious Diseases 1991;164:757-60 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6404-0019$01.00
transmitted disease clinics or physician practices in the United States. Effective antiviral chemotherapy for genital herpes now exists and efficient laboratory tests are needed for distinguishing ulcers due to HSV from those associated with other infections. such as Treponema pallidum and Haemophilus ducreyi or other noninfectious causes [I, 2]. Previous studies have shown that stage of clinical infection (first vs. reactivation episode), clinical stage of the lesion (vesicular vs. ulcerative lesion), and host immune status (immunocompetent vs. immunosuppressed patients) influence the ability to isolate HSV from genital lesions [3-5]. Most studies have shown that among immunocompetent persons with recurrent genital herpes, HSV can be isolated from a single lesion swab in only 40%-70% of such samples. Subsequent
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I. Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, Schaeffer JJ. Selectivity of action of an antiherpetic agent. 9-(2-hydroxyethoxymethyl)guanine. Proc Natl Acad Sci USA 1977;74:5716-20. 2. Furman PA, St. Clair MH, Fyfe JA, Rideout JL, Keller PM, Elion GB. Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate. J Virol 1979;32:72-7. 3. Elion GB. Mechanism of action and selectivity of acyclovir. Am J Med 1982;73:7-13. 4. Clive D, Turner NT, Hozier J, Batson AG, Tucker WE Jr. Preclinical toxicology studies with acyclovir: genetic toxicity tests. Fundam Appl Toxicol 1983;3:587-602. 5. Tucker WE Jr, Macklin A W, Szot RJ, et al. Preclinical toxicology studies with acyclovir: acute and subchronic tests. Fundam Appl Toxicol 1983;3:573-8.
Concise Communications
5. Seidel-Dugan C, Ponce de Leon M, Friedman HM, Eisenberg RJ, Cohen GH. Identification of C3b-binding regions on herpes simplex type 2 glycoprotein C. J Virol 1990;64: 1897-906. 6. Friedman HM, Vee A Digglemann H, et al. Use ofa glucocorticoid-inducible promoter for expression of herpes simplex type I glycoprotein gC I, a cytotoxic protein in mammalian cells. Mol Cell Bioi 1989;9:2303-14. 7. Seidel-Dugan C, Ponce de Leon M, Friedman HM, et al. C3b receptor activity on transfected cells expressing glycoprotein C of herpes simplex virus type I and 2. J Viral 1988;62:4027-36. 8. Smiley ML, Hoxie lA, Friedman HM. Herpes simplex virus type I infection of endothelial, epithelial, and fibroblast cells induces a receptor for C3b. J Immunol 1985; 134:2673-8.
10. Smiley ML, Friedman HM. Binding of complement component C3b to glycoprotein C is modulated by sialic acid on herpes simplex virus type I infected cells. J Virol 1985;55:857-61. II. Devereux JR, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 1984; 12:387-95. 12. Corbi AL, Kishimoto TK, Miller LJ, Springer T A. The human leukocyte adhesion glycoprotein Mac-I (complement receptor type 3, CDllb) ll' subunit. J Bioi Chem 1988;263:12403-1 I. 13. van Strijp JAG, van Kessel KPM, van der Tol ME, Verhoef J. Complement-mediated phagocytosis of herpes simplex virus by granulocytes. J Clin Invest 1989;84: 107-12. 14. Harold BC, WuDunn 0, Soltys N, Spear PG. Glycoprotein C of herpes simplex virus type I plays a principal role in the adsorption of virus to cells and in infectivity. J Virol 1991;65: 1090-8. 15. Langeland N, Oyan AM, Marsden HS, et al. Localization on the herpes simplex virus type I genome of a region encoding proteins involved in adsorption to the cellular receptor. J Virol 1990;64: 1271-7.
A Double-Blind, Placebo-Controlled Cytogenetic Study of Oral Acyclovir in Patients with Recurrent Genital Herpes Donald Clive, Lawrence Corey, Richard C. Reichman, L. Gray Davis, and John C. Hozier
Burroughs Wellcome, Research Triangle Park. North Carolina; Departments of Laboratory Medicine and Medicine, University of Washington, Seattle; Infectious Diseases Unit. University of Rochester School of Medicine, New York; Applied Genetics Laboratory, Melbourne, Florida
The antiherpes drug acyclovir breaks chromosomes in vitro at millimolar concentrations and at highly toxic doses in rodents but does not induce single-gene mutations. Recurrent genital herpes patients were examined to determine if such chromosomal damage occurs in peripheral lymphocytes during acute or chronic acyclovir therapy. Patients were randomly assigned to receive acyclovir suppressively and for recurrences, placebo suppressively and acyclovir for recurrences, or placebo suppressively and for recurrences (n ~ 20 for each group; all treatment doubleblind). Normal volunteers and acyclovir-treated cultures served as additional controls. Cytogenetic analyses were done at enrollment (pretreatment), on day 5 of acute acyclovir or placebo treatment for the first postenrollment recurrence (postacute), and at the end of a year on study (postchronic). Cells in metaphase, 150 for each patient, were examined at each time point for structural and numerical chromosomal abnormalities. No cytogenetic effects of chronic or acute oral acyclovir treatment were found relative to lifestyle controls, pretreatment controls, or placebo treatment.
Acyclovir (ACV; 9-(2-hydroxyethoxymethyl)guanine; molecular weight, 225.2), a potent inhibitor of herpes simplex virus types I and 2 (HSV-I, -2), has extremely low toxicity
Received 4 February 1991; revised 23 May 1991. Presented in part: Environmental Mutagen Society meeting. Charleston. South Carolina. March 1988 (abstract 55). Informed consent was obtained from patients or guardians, and guidelines for human experimentation of the ethical committees of the University of Rochester School of Medicine and University of Washington Department of Laboratory Medicine were followed. Grant support: National Institutes of Health (AI-2038I ). Reprints or correspondence: Dr. Donald Clive. Burroughs Wellcome Co., Research Triangle Park, NC 27709. The Journal of Infectious Diseases 1991;164:753-7 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6404-0018$01.00
for uninfected host cells [I, 2]. This high therapeutic index results from the specific activation of ACV to its monophosphate by a herpes-specified thymidine kinase; in herpes-infected cells, this can subsequently be converted to the di- and triphosphates by normal cellular enzymes [3]. ACV triphosphate acts by preferential inhibition of the herpes-specified DNA polymerase [3]. The genetic toxicology of ACV in vitro and in rodent in vivo studies has been presented elsewhere [4]. ACV does not induce single-gene mutations in a variety of in vitro microbial or mammalian cell systems at concentrations up to and often exceeding 2500 ,ttg/ml but is clastogenic at concentrations > 125 .tLg/ml in vitro or the equivalent plasma levels in vivo. Such high plasma levels crystallize out in renal tubules and lead to extensive obstructive nephropathy [5]. Although
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9. Gaither TA, HammerCH, Frank MM. Studies on the molecularmechanisms ofC3b inactivation and a simplified assay for b IH and the C3b inactivator (C3nINA). J Immunol 1979; 123: 1195-233.
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typically lethal to about one-third of test animals within 2-4 days of single intravenous dose administration, this highdose effect is masked by the typical sacrifice schedule of most in vivo short-term studies. This specificity for inducing chromosomal damage formed the basis for investigating the genotoxicity of ACV in humans in a cytogenetic study. The present investigation was part of a double-blind, placebocontrolled study of the ability of oral acyclovir to suppress the frequency of recurrent genital herpes outbreaks [6].
Patients and Methods
To ensure blind scoring, this blood specimen was assigned a number previously deleted from the clinical sequence and was timed to "arrive" at the cytogenetics laboratory at the appropriate time. Experimental design method for the control ofbias. Enrolled patients were assigned a sequential patient number that randomly and blindly placed them into one of the principal treatment groups. Patients were treated with ACV or placebo suppressively and during each recurrence according to their group assignment. In addition, non-herpes infected volunteers were selected at each clinical center and assigned to group IV. This last group received no drug and served as matched lifestyle controls for the incidence of chromosome damage. At least 20 patients were enrolled in each group. Each blood sample was assigned a unique and sequential specimen number. This number was the only information accompanying the blood sample when cytogenetic analyses were done. Codes were broken at Burroughs Wellcome Genetic Toxicology Laboratory only after the data had been transmitted and recorded. Patients were screened for occupation, recent viral diseases, use of social drugs, medications, smoking, and other factors likely to influence the outcome of the study (table 1). Cytogenetics. Each blood specimen was cultured in quadruplicate and prepared for cytogenetic analysis in duplicate. The medium used was RPMI 1640 supplemented with 20% fetal calf serum, 100 units/rnl penicillin, 100 Ilg/ml streptomycin, and 2 mM L-glutamine. The following were added to 4.7-ml aliquots of this freshly supplemented medium in each of four T-25 flasks: 0.3 ml of heparinized blood, one drop of heparin (at 1000 units/ ml), and 200 III of freshly reconstituted phytohemagglutinin (M-Form; GIBCa, Grand Island, NY). Cultures were incubated at 37°C for 42-48 h. Three hours before the end of the culture (groups I-IV) or treatment (group V) period, the quadruplicate cultures were reTable 1.
Demographics for patients treated orally with acyclovir. Group
Age Mean ± SD Range Sex Male Female Race White Other Smoking Yes No Not ascertained Oral contraceptives (females only) Use Don't use
II
III
IV
32 ± 7 22-50
34 ± 10 22-62
32 ± 6 23-43
31 ± 5 23-42
17 (63) 10 (37)
18 (62) II (38)
15(62) 9 (38)
12 (48) 13(52)
25 (93) 2 (7)
28 (97) I (3)
23 (96) I (4)
24 (96) I (4)
II (41) IS (56) I (4)
9 (31) II (38) 9 (31)
5 (21) 12 (50) 7 (29)
9 (36) 15 (60) I (4)
5 (50) 5 (50)
3 (27) 8 (73)
3 (33) 6 (67)
I (8) 12 (92)
NOTE. Groups: I, acyclovir suppressively andfor recurren~es; II, placebo suppressively and acyclovir for recurrences; III, placebo ~uppresslvely andfor recurrences; IV, lifestyle controls. Data are no, (%) unless indicated.
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Test substances. ACV capsules (200 mg; Burroughs Wellcome) were used to treat enrolled patients. ACV was also used to dose blood cultures set up for positive controls. For placebo control, lactose in capsule formulation identical with that of ACV was used. Subjects. Subjects were those enrolled in a placebo-controlled study (protocol 137) in 1984-1987 that compared suppressive and acute oral ACV therapy in the management of recurrent genital herpes [6]. This study was done at two centers: the University of Washington and the University of Rochester School of Medicine. Patients diagnosed with culture-documented recurrent genital herpes were enrolled in this study at a time when they were asymptomatic. On enrollment they were randomized into one of three treatment groups: group I, 400 mg of ACV twice daily suppressively (i.e., throughout the l-year duration of the study or until patients dropped out) and 200 mg of ACV five times daily acutely (i.e., for 5 days during each outbreak) for recurrences; group II, placebo suppressively and ACV for recurrences; and group III, placebo suppressively and for recurrences. In addition, non-herpes infected volunteers were selected at each clinical center to serve as lifestyle controls (group IV). The lifestyle controls were confirmed as antibody-negative to HSV-I and -2. These volunteers were of similar age to groups I-III and were selected from personnel within the clinical laboratories at the two medical centers. They received no treatment and were on no other therapy. An in vitro positive control group (group V) was produced at the cytogenetic laboratory (see below). Blood specimens. The first of three blood specimens for cytogenetic analysis was drawn at enrollment and served to determine each patient's pretreatment (i.e., control) level of cytogenetic damage. A second specimen was collected on day 5 of acute ACV or placebo treatment for the first postenrollment recurrence and served to measure cytogenetic effects of this acute ACV treatment. The third and final specimen was collected at the end ofa year on study and served to measure cumulative cytogenetic effects ofa chronic suppressive therapy. Each specimen consisted of 10 ml of blood drawn into a Venoject or Vacutainer tube containing sodium heparin. Each blood specimen was shipped overnight from the two clinical laboratories to the laboratory performing the cytogenetic analysis (Applied Genetics Laboratories). For group V positive controls, blood was drawn at predetermined intervals from a volunteer at Applied Genetics Laboratories, cultured, and treated with ACV ( 175 Ilg/ ml for 24 h) by a person not involved in the cytogenetic analysis.
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Results Table I summarizes the demographic characteristics of each treatment and control group. No significant differences were detected for age, sex, race, smoking, or incidence of recent viral illnesses. Somewhat fewer women used oral contraceptives in the lifestyle control group than in the three treatment groups. Although there was significant nonascer-
tainment for some parameters, this is not believed to have led to any bias that might have affected the outcome of this study. Table 2 summarizes, for each group of patients or controls, the cytogenetic aberration results at the time of enrollment, after treatment for the first recurrence on study, and after I year of therapy. No statistically significant differences in number of chromosomal aberrations over time or between placebo and any of the ACY recipients were noted. Similarly, the frequencies ofchromosomal aberrations were equal in the lifestyle controls and all treatment groups. No patient exhibited >6.3 chromosome abnormalitiesflOO cells. No statistically significant differences in mitotic indices were noted over time, between placebo and any group of ACV recipients, or between virus-infected and uninfected controls. Table 3 summarizes the incidences of numerical chromosome damage (hypodiploidy, hyperdiploidy, and polyploidy) by treatment group and time. There were no significant differences among groups except for an increase in hyperdiploidy and a possible reduction in hypodiploidy in group V (positive controls). The latter may reflect the fresher, untraveled blood specimens used in this group. Group V consisted of seven positive control specimens treated in vitro with ACY. The mean (±SD) percentage of aberrant cells in this group was 6.7 ± 2.9; the mean (±SD) number of aberrations per 100 cells was 8.4 ± 3.9. This reflects the expected in vitro effects of ACY as described previously [4]. Overall, the incidence of structural and numerical chromosome alterations was not significantly affected by chronic or acute ACY treatment relative to either enrollment values,
Table 2. Lack of chromosome breakage after acute and chronic acyclovir treatment. % aberrant cells Group, time I, Pretreatment
Postacute Postchronic II, Pretreatment Postacute Postchronic III. Pretreatment Postacute Postchronic IV, Lifestyle controls V, Positive controls
n
27 19 24 28 27 17 24 22 15 25 7
Mean ± SO 0.8 0.8 0.6 1.2 1.2 0.5 0.8 1.0 0.8 0.8 6.7
± ± ± ± ± ± ± ± ± ± ±
0.8 1.1 0.6 1.3 l.l 0.5 1.0 1.2 0.6 1.0 2.9
No. aberrations/ 100 cells Range
Mean ± SO
Range
0.0-2.7 0.0-4.7 0.0-2.0 0.0-5.7 0.0-4.2 0.0-1.3 0.0-3.3 0.0-4.7 0.0-2.0 0.0-3.3 3.3-10.7
0.9 ± 0.8 0.9± 1.1 0.6 ± 0.6 1.2 ± 1.3 1.4 ± 1.6 0.5 ± 0.5 1.0 ± 1.3 1.3 ± 1.8 0.8 ± 0.6 0.9±1.1 8.4 ± 3.9
0.0-3.3 0.0-4.7 0.0-2.0 0.0-5.7 0.0-6.3 0.0-1.3 0.0-4.0 0.0-6.3 0.0-2.0 0.0-3.3 4.0-13.3
Mitotic indices, Mean ± SO 1.6 ± 1.2 1.8 ± 1.5 1.5 ± 1.0 1.5±1.1 1.3 ± 1.0 1.4 ± 0.8 1.6 ± 1.8 1.3±1.1 1.5 ± 0.9 2.1±1.9
NOTE. Groups: I, acyclovir (400 mg orally twice daily for I year) suppressivelyand acyclovir (200 mg orally five times daily for 5 days) for recurrences; 11. placebo suppressivelyand acyclovir for recurrences; III, placebo suppressively and for recurrences; IV. laboratory personnel from the two centers; V. blood from normal volunteers treated in vitro with acyclovir (175 ltg/mi. 24 h). Pretreatment blood was sampled at enrollment: postacute, day 5 of treatment for first postenrollment recurrence; postchronic, at end of I year on study. Actual number of days on study (±SD): group 1,365 ± 82; group II. 327 ± 64; group III, 325 ± 88. Lower number of patients after first recurrence is due to dropouts and to absence of recurrences in some patients; lower number at end of study is due to dropouts.
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duced to duplicates by combining in pairs; these duplicate preparations were processed separately thereafter. Demecolcine was added to each flask to a final concentration of 0.2 J,Lg/ml and incubation continued. After 3 h of mitotic arrest, the cells were harvested using 0.075 M KCI and 3: I methanol-glacial acetic acid fixative, dropped onto clean slides, and air-dried overnight. Slides were stained with 2% Giemsa stain in phosphate buffer. One hundred fifty cells in metaphase were examined for each culture. Structural and numerical chromosomal abnormalities and microscope stage vernier coordinates for each metaphase were recorded. The mitotic index (percentage of cells undergoing mitosis, based on 1000 cells counted) was also recorded for each culture. For classification of cytogenetic aberrations, the following structural and numerical chromosomal abnormalities were characterized: chromosome gains and losses, chromatid-type aberrations, chromosome-type aberrations, and severely damaged cells (;;::'10 scorable aberrations). Chromatid and chromosome-type aberrations were further subdivided into deletions and interchanges. By convention, chromatid and chromosome gaps and pulverized chromosomes and cells were recorded but not included in the aberration analysis. Statistical analysis. Kruskal-Wallis tests were used to test for differences between treatment groups and for effects within each group resulting from acute or chronic treatment.
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Table 3.
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Lack of numerical chromosome effects after acute and chronic acyclovir treatment. % Numerically abnormal cells
Group. time
NOTE.
Hypodiploid
27 19 24 28 27 17 24 22 IS 25 7
14.1 ± 6.9 15.6 ± 14.1 10.2±4.5 15.1 ± 8.4 13.3 ± 9.7 10.0 ± 7.3 14.2 ± 7.0 19.4 ± 20.3 10.8 ± 4.8 12.3 ± 6.6 6.9 ± 3.0
Hyperdiploid 0.2 ± 0.5 ± 0.6 ± 0.4 ± 0.4 ± 0.6 ± 0.3 ± 0.6 ± 0.5 ± 0.6 ± 1.6 ±
0.5 0.5 0.9 0.6 0.7 1.1 0.4 0.9 0.6 0.7 0.8
Polyploid 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
± 0.4
± 0.0 ± 0.0 ± 0.0
± 0.0 ± 0.0
± 0.0 ± 0.2 ± 0.0 ± 0.0
± 0.0
See footnote to table 2.
equivalent placebo control group values, or lifestyle control values.
Discussion Analysis of structural and numerical alterations and group mitotic indices showed no effect of chronic or acute oral ACV treatment in recurrent genital herpes patients relative to lifestyle controls, pretreatment controls, or placebo treatment. It is instructive to consider what factors might be involved in this lack of response. The simplest and most obvious explanation for these negative effects is that the mean peak plasma levels of ACV (0.83 and 1.61 JLg/ml after 200- and 600-mg doses, respectively, six times daily) were too low to exert any clastogenicity, particularly in light of the threshold effect seen at ,..." 125 JLg/ml in the in vitro cytogenetic study [4]. However, this clastogenicity of ACV only at high (i.e., millimolar) concentrations in vitro or at comparably high plasma levels in vivo in non-herpes infected systems [4] may not be relevant to the clinical situation, since it presumably proceeds by conversion of ACV to the monophosphate metabolite by reversing the activity of a cellular 5'-nucleotidase. It could be argued that therapeutic levels ofACV in conjunction with the far more efficient (i.e., at micromolar levels) HSV thymidine kinase within an infected cell, followed by cell contact-mediated transport of the phosphorylated ACV from these cells to target peripheral lymphocytes, might cause an increase in chromosomal damage in the latter. However, this possibility receives no support from the present study (tables 2 and 3). Other aspects of the clinical situation might be anticipated to induce clastogenic effects. For instance, an increase in cytogenetic damage might be anticipated as a result of the
viral infection itself, for a number of reasons. Viruses per se are clastogenic [7]; also, untreated patients infected with HSY-l or -2 have been reported to have elevated levels of sister-chromatid exchanges (SCEs) [8], and SCEs are often correlated with the more traditional and genetically better understood chromosomal aberrations measured in this study. In addition, certain types of behavioral stress are genotoxic in rodents [9-11], and these recurrent genital herpes patients may be stressed. It is possible that the above-mentioned increase in SCEs seen in HSV-infected patients resulted from this associated stress rather than from a direct effect of the virus on the genome. No indication of cytogenetic damage was seen in the pretreatment samples compared with those of the lifestyle controls (1. 1 ± 1.2 aberrations/ 100 cells for pooled recurrent genital herpes patients from groups I-III [n = 79] vs. 0.8 ± 1.1 aberrations/ 100 cells for HSV-free lifestyle controls [n = 25]). Thus, for these patients, neither the viral infection nor the stress that might be associated with it were capable of inflicting measurable cytogenetic damage in peripheral lymphocytes. Finally, if ACV (or simply being enrolled in this study) suppresses recurrences, their associated stress, and hence their clastogenicity, it might be argued that this reduction could be masking an equal induction of chromosome breakage by ACV. This is unlikely for two reasons: first, chromosome damage in peripheral lymphocytes persists much longer than the duration of this study, so that many of the virus- (and possibly stress-) induced cytogenetic lesions would not disappear, and any ACY-induced damage would be additive; second, as mentioned above, there was no evidence that such a virus- or stress-related chromosomal effect existed in these patients at entry. In conclusion, this study failed to demonstrate (under conditions that detected an in vitro effect) any significant effect of either acute or chronic oral therapy with ACV on the inci-
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I. Pretreatment Postacute Postchronic II. Pretreatment Postacute Postchronic III, Pretreatment Postacute Postchronic IV, Lifestyle controls V, Positive controls
n
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dence of structural or numerical chromosome damage in peripheral lymphocytes of recurrent genital herpes patients.
6. Mattison HR, Reichman RC, Benedetti J, et al. Double-blind, placebocontrolled trial comparing long-term suppressive with short-term oral acyclovir therapy for management of recurrent genital herpes. Am J Med 1988;85:20-5.
References
7. Gershenson SM. Viruses as environmental mutagenic factors. Mutation Res 1986;167:203-13. 8. Ghosh R, Ghosh PK. Sister-chromatid exchanges in herpes simplex infection. Mutation Res 1983; 119:303-8. 9. Seredenin SB, Durnev AD, Vedernikov AA. Effect of emotional stress on the frequency ofchromosomal aberrations in mouse bone marrow cells. Bull Exp Bioi Med 1980;89:972-3. 10. Fischman HK, Kelly DD, and Rainer JD. Behavioral stress and sister chromatid exchanges [abstract]. Environ Mol Mutagen 1985; 7( suppl 3): 51. II. Kelly DD, Pero R W, Fischman HK. Stress-induced chromosomal alterations: sister chromatid exchanges and unscheduled DNA synthesis [abstract 198.11]. In: Abstracts, 15th annual meeting, Society for Neuroscience (Dallas). Washington, DC: Society for Neuroscience, 1985; I 1:664.
Extended Duration of Herpes Simplex Virus DNA in Genital Lesions Detected by the Polymerase Chain Reaction Richard W. Cone, Ann C. Hobson, Janet Palmer, Michael Remington, and Lawrence Corey
Departments of Laboratory Medicine and Medicine, University of Washington, Seattle
To evaluate the utility of the polymerase chain reaction (PCR) for documenting herpes simplex virus (HSV) in persons with reactivated genital lesions viral isolation was compared with a recently developed PCR method. Three women experiencing four episodes of recurrent genital herpes were followed for 10 days per episode with daily examination and duplicate swabs of the lesions, one for HSV culture and one for PCR. HSV type 2 was cultured from three of four episodes and the mean duration of viral isolation from recurrent genital lesions was 2.6 days. PCR detected HSV DNA from lesion swabs during all four episodes, and HSV DNA was positive for an average of 6.8 days. HSV DNA was demonstrated in ulcerative lesions on 15 of 17 days versus 3 of 17 days by viral isolation (P < .01). HSV PCR became negative when the lesions reepithelialized. These data suggest that PCR is a more sensitive measure of HSV infection than routine viral culture and that PCR detects the presence ofHSV at times when culture is negative.
Herpes simplex virus (HSV) infection is the most common cause of genital ulcerations among patients visiting sexually
Received 12 March 1991; revised 28 May 1991. Presented in part: 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, October 1990 (abstract 199). Informed consent was obtained from the patients studied, and human experimentation guidelines of the University of Washington were followed in the conduct of this research. Grant support: National Institutes of Health (AI-20381). Reprints or correspondence: Dr. Richard Cone, Children's Hospital and Medical Center. 4800 Sand Point Way N.E., Room D-536, Seattle, WA 98105.
The Journal of Infectious Diseases 1991;164:757-60 © 1991 by The University of Chicago. All rights reserved. 0022-1899/91/6404-0019$01.00
transmitted disease clinics or physician practices in the United States. Effective antiviral chemotherapy for genital herpes now exists and efficient laboratory tests are needed for distinguishing ulcers due to HSV from those associated with other infections. such as Treponema pallidum and Haemophilus ducreyi or other noninfectious causes [I, 2]. Previous studies have shown that stage of clinical infection (first vs. reactivation episode), clinical stage of the lesion (vesicular vs. ulcerative lesion), and host immune status (immunocompetent vs. immunosuppressed patients) influence the ability to isolate HSV from genital lesions [3-5]. Most studies have shown that among immunocompetent persons with recurrent genital herpes, HSV can be isolated from a single lesion swab in only 40%-70% of such samples. Subsequent
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I. Elion GB, Furman PA, Fyfe JA, de Miranda P, Beauchamp L, Schaeffer JJ. Selectivity of action of an antiherpetic agent. 9-(2-hydroxyethoxymethyl)guanine. Proc Natl Acad Sci USA 1977;74:5716-20. 2. Furman PA, St. Clair MH, Fyfe JA, Rideout JL, Keller PM, Elion GB. Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate. J Virol 1979;32:72-7. 3. Elion GB. Mechanism of action and selectivity of acyclovir. Am J Med 1982;73:7-13. 4. Clive D, Turner NT, Hozier J, Batson AG, Tucker WE Jr. Preclinical toxicology studies with acyclovir: genetic toxicity tests. Fundam Appl Toxicol 1983;3:587-602. 5. Tucker WE Jr, Macklin A W, Szot RJ, et al. Preclinical toxicology studies with acyclovir: acute and subchronic tests. Fundam Appl Toxicol 1983;3:573-8.
