Type Incidence of Herpes Simplex Virus in Clinical Isolates from Patients with Herpes Genitalis

Abstract

Original ArticlesType Incidence of Herpes Simplex Virus in Clinical Isolates from Patients with Herpes Genitalis Abd Ul-Ghani Mohamed Al-Samarai, MB, ChB, M Med Sci, PhD Anisa Abd Ul-Habib Shareef, BSC, M Med Sci George Kinghorn, and MB, ChB, MRCP, MD Christopher PotterBSC, MRCPath, PhD Abd Ul-Ghani Mohamed Al-Samarai Address correspondence and reprint requests to Dr. Al-Samarai: Department of Microbiology, Basrah Medical College, Basrah, Iraq. From the Departments of Microbiology and Anatomy and Cell Biology, Basrah Medical College, Basrah, Iraq Search for more papers by this author , Anisa Abd Ul-Habib Shareef From the Departments of Microbiology and Anatomy and Cell Biology, Basrah Medical College, Basrah, Iraq Search for more papers by this author , George Kinghorn From the Departments of Genitourinary Medicine and Virology, Sheffield University Medical School, Sheffield, UK Search for more papers by this author , and Christopher Potter From the Departments of Genitourinary Medicine and Virology, Sheffield University Medical School, Sheffield, UK Search for more papers by this author Published Online:1 Mar 1990https://doi.org/10.5144/0256-4947.1990.156SectionsPDF ToolsAdd to favoritesDownload citationTrack citations ShareShare onFacebookTwitterLinked InRedditEmail AboutAbstractIsolates from a total of 228 patients with clinical herpes simplex were included in this study: 92 were from patients in their first episode and 136 were from patients with recurrent genital herpes. All isolates were typed by enzyme-linked immunosorbent assay. In the first episode group, 52 (56.5%) of the isolates were shown to be herpes simplex virus type 1 (HSV-1) and 40 (43.5%) to be type 2 (HSV-2). In patients with recurrent genital herpes, HSV-1 was isolated from 10 (7.3%), HSV-2 from 121 (89%), and both types from five (3.7%). There is a significant difference in the incidence of HSV-1 between the initial and recurrent herpes infection (P < 0.0001). The high incidence of HSV-1 in the initial episode may be due to oro-génital contact. The above findings have a possible implication in the development of drugs and vaccines in the prevention of herpes genitalis. HSV-1 may also be shown to play a role in the etiology of cervical neoplasia.IntroductionHerpes simplex virus (HSV) infection of the genitalia is a sexually transmitted disease that is increasing in incidence.1,2 Initial exposure to the virus usually produces acute symptoms and severe discomfort, but the disease may also be asymptomatic. After the first episode of infection, the virus becomes latent in the sensory ganglia and patients remain at risk for recurrent herpes.Herpesvirus hominis is divided into two types: type 1 (HSV-1) and type 2 (HSV-2), distinguished by serologic, biologic, or antigenic criteria. It is generally accepted that there is a difference in the site of involvement and mode of transmission for the two serotypes.3-5 HSV-1 produces nongenital infections and HSV-2 has been shown to be the major causative agent in genital and neonatal infections.4 HSV-2 accounts for 89% of the uro-genital infections in females and 97% in males.6 Other reports, however, have indicated that HSV-1 may play a greater part in genital infections than was previously thought. Smith et al7 found that 22% of the genital isolates from females and 9% from males are HSV-1 in the Edinburgh, Scotland, population. In the U.S., Corey et al8 isolated HSV-1 from genital lesions in 7% of their patients in the first episode of the disease and in only 2% of those with recurrent genital herpes. Kaufman et al,9 however, isolated HSV-1 from 13.4% of their patients with recurrent disease.Thus HSV-1 has accounted for a widely different proportion of isolates from patients with first episodes of genital herpes, averaging 7% in Seattle, Washington, U.S., 30% in Northern Ireland, 43% in Japan, and 63% in Sheffield, England.2,8,10,11 We report here the incidence of HSV-1 and HSV-2 in patients with first episode and recurrent genital HSV infection in Sheffield, England.MATERIAL AND METHODSTissue CultureVero cells (Flow Laboratories, Irvine, Scotland) were grown as monolayers in 199 medium containing 10% (v/v) fetal calf serum. Cells were subcultured at a regular interval of 3 days using a split of 1:4.VirusesHSV-1, strain F (obtained from Dr. B. Roizman, Chicago, Illinois, U.S.), and HSV-2, strain 333 (obtained from Dr. F. Rapp, Milton Hershey Medical Center, Hershey, Pennsylvania, U.S.), were propagated at a multiplicity of 0.1 to 1 plaque-forming unit per cell in Vero cells.Isolation of Viruses from Clinical SpecimensSwabs were used to obtain vesicular fluid or specimens from ulcerated lesions of the genitalia or cervix in women and urethra in men and these specimens were used for virus isolation. Upon receipt in the laboratory, the specimens were placed immediately into 2 ml of transport medium; the specimens were then divided into three aliquots and stored at -80°C until tissue cultures were available.The viruses were isolated and stocks prepared and titrated as described previously.11Identification of HSV IsolatesViruses isolated from the clinical specimens were typed using an enzyme-linked immunosorbent assay (ELISA) described by Vestergaard and Jensen.12 Specific antibodies prepared in rabbit serum against HSV-1 and HSV-2 were diluted in coating buffer and 0.1 ml of the preparation inoculated into wells of microtest plates. The plates were left at room temperature. After incubation, the contents of the wells were decanted and plates washed for 3 minutes with PBS-Tween; this was repeated three times. The 100 μl of reference antigens (HSV-1, strain F; HSV-2, strain 333) or clinical isolates, prepared in a series of two-fold dilution in PBS/1% triton-X-100, were each added to a duplicate row of wells; one row was coated with anti-HSV-1 and the second row with anti-HSV-2 antibodies. The plates were incubated at 4°C overnight. The plates then were washed as described above and 100 μlof rabbit anti-HSV-1 or rabbit anti-HSV-2 antibody conjugated to peroxidase was added to wells coated with type-specific antibody for HSV-1 and HSV-2, respectively. These reagents were diluted 1:200 in PBS-Tween + 1% bovine serum albumin (BSA). The plates were then incubated for 1 hour at 37°C. After washing as described above and once with substrate buffer, 100 μl of substrate was dispensed into each well and plates incubated for 10 to 30 minutes at room temperature. The reaction was then stopped by the addition of 50 μl of 2N H2SO4 per well. The results were read in a spectrophotometer at 490 nm.The typing was checked by ELISA utilizing monoclonal antibody, as described by Nilheden et al.13 Aliquots (200 ml) of Vero cell suspension were seeded into the wells of a flat-bottomed microplate and incubated overnight at 37°C in a humidified atmosphere. The wells were then emptied and 200 μl of undiluted virus isolate added to each of four wells. The plates were incubated overnight in a carbon dioxide incubator. The infected cells were fixed with 0.2% glutaral-dehyde (100 μl) for 1 hour and then washed with three changes of PBS-Tween. One hundred microliters of monoclonal antibodies (B1C1 & C4D5) diluted in PBS-Tween with 1% BSA was then added (two wells for each) and the plates further incubated for 1 hour at 37°C and washed as described above. Then 100 μl of antimouse IgG conjugated to peroxidase was added to each well at a dilution of 1:200. One hundred microliters of substrate was finally added to the wells and plates and incubated for 10 minutes. Results could be read visually or by using a spectrophotometer at 490 nm. Four wells on each plate were used as controls, and HSV-1 and HSV-2 were used as reference antigens in these controls.PatientsThe clinical specimens were obtained from patients with either their first episode of HSV genital infection or a recurrent infection. Swabs were used to take material from external lesions, the endocervix of women, and the urethra of men. First episodes of genital HSV were classified into two groups: (1) true primary infection, occurring in a patient who has had no previous HSV infection as evidenced by lack of serum antibody to both virus types, and (2) nonprimary first episodes in which the first symptomatic genital infection occurs in a patient who already has HSV antibody.8Specimens were collected from 92 patients in the first episode of genital HSV infection. Of these, 62 were female and 30 were male. The mean age was 22.6 (range, 18 to 38) years. Of 92 patients, 75 (82%) had true primary infection. For patients with recurrent infection, HSV was isolated from 136 patients with a mean age of 28.8 (range, 18 to 50) years; 64 were male and 72 were female.RESULTSA total of 92 HSV clinical isolates obtained from male and female patients in their first episode of genital HSV infection were typed. The results are shown in Table 1. Fifty-two (56.5%) of the isolates were HSV-1, as typed by ELISA. HSV-1 was more common in females (64.5%) than in males (40%) but was the reverse for HSV-2.Table 1. Incidence of HSV type in genital herpes infection.Table 1. Incidence of HSV type in genital herpes infection.A total of 136 isolates were typed in the patients with recurrent HSV infection. Ten (7.3%) of the isolates were HSV-1. Both HSV-1 and HSV-2 were isolated from five (3.7%) patients (Table 1). Most of the isolates from both men and women with recurrent genital HSV infection were type 2, thus the incidence of HSV-1 in the first episode (56.5%) was significantly less (P < 0.0001) than that in recurrent infection (7.3%).The findings were analyzed according to true primary and nonprimary first episodes, and the results are shown in Table 2. Patients with true primary infections mostly had type 1 virus. In non-primary and recurrent genital HSV infections most of the isolates were HSV-2. Each type showed an opposite incidence trend (Table 2). No apparent correlation was shown between the causative virus type and the nature of the genital lesion, but there was a significant correlation between the virus type and frequency of recurrence.Table 2. Incidence of HSV type in relation to infection type.Table 2. Incidence of HSV type in relation to infection type.DISCUSSIONGenital HSV infection is an important sexually transmitted disease that has increased in incidence in recent years. Reports of patients with genital herpes have suggested that it is not only the cause of physical disability but also produces psychological consequences that can be even more distressing and serious.14 There is also a close association between HSV infections and cervical neoplasia.15 The results of the present study and those of Corey et al8 do not confirm the association of genital herpes with the HSV-2 serotype. HSV-1 was isolated more frequently in our patient population than has been reported from other places.2,6-10In the present study, 56.5% of the isolates from patients (both males and females) in their first episode were HSV-1 and 43.5% contained HSV-2. This confirms the findings reported in a a small group of patients from Sheffield, England.11 However, we found HSV-2 was more frequently isolated from cases of recurrent genital HSV infection than first episodes, so there is need for differentiating the incidence of HSV-1 according to the clinical manifestation (i.e., true primary, non-primary first episode, and recurrent infection), as shown in Table 2. HSV-1 was isolated from 60% of the patients with true primary disease compared to 41% of the patients with nonprimary first episode. The reduced incidence of HSV-1 in patients with nonprimary first episode suggests that previous oral HSV-1 infection protects against the acquisition of genital HSV-1 infection.8It is unknown whether prior HSV-1 infection also protects against genital HSV-2 infection. Thus it is concluded that there is a significant difference (X2 = 64.53; P < 0.0001) in the type incidence of HSV between the initial and recurrent infection.The differences in incidence of HSV-1 and HSV-2 between initial and recurrent infections may be due to the lower rate of recurrence of genital type 1 infection compared to genital type 2 infection.16 The difference in the recurrence rate between types 1 and 2 could be explained by the decreased latency of HSV-1 in the sacral root ganglia compared to HSV-2. This difference between the two virus types may stem from the immunosuppressive ability of HSV-2 that was recently reported.5The increased incidence of HSV-1 in patients in the first episode of infection is due to more frequent oral-genital contact.8,11 The incidence of HSV-1 in our study is higher than that reported for Northern Ireland. In the present study, 40% of the men and 64.5% of the women with initial infection had HSV-1 whereas in the study from Northern Ireland 60% of the men and 35.5% of the women had HSV-2 infection. In contrast, HSV-1 was isolated from 23.5% of men with genital herpes and 41.3% of women in a study from Northern Ireland conducted during 1982 to 1984. In a study from Edinburgh, Scotland, carried out during 1977 to 1979 these figures were 16% for men and 32% for women. The incidence of HSV-1 for the first episode was higher in females than in males and this may be due to the fact that oral-genital sex is practiced more by females than by males, or it may be due to anatomic variations that may increase the exposure area. Thus the typing of the causative virus is of prognostic importance, and this confirms the conclusions of other reports.10,11Our data agree with that from the Sheffield population11 concerning the HSV-1 incidence in the first episode, but is in conflict with their finding of an increased incidence in HSV-1 in cases of recurrent infection as well.The high rate of HSV-1 isolation in the present study has also been reported recently in another study.2 As mentioned previously, HSV-2 has been shown to be associated with cervical neoplasia,18,19 and in a recent report, four of five patients with cervical atypia had herpes genitalis caused by HSV-1, which suggests that HSV-1 may play a role in the etiology of cervical neoplasia— perhaps as an initiator.20 It is also important to take this finding into account when an HSV vaccine is produced for the prevention and control of herpes genitalis. Herpes simplex serotypes show different sensitivities to antiviral agents. It is therefore important to refer to the virus type of a strain tested for drug sensitivity.Finally, 18% of the patients with initial genital HSV infection had serologic evidence of previous HSV infection, and 82% had true primary infection. This percentage of true primary infection is higher than that reported from Seattle, Washington, in the United States.8 The higher proportion of patients with true primary genital herpes reflects the particular interest and vigilance in the diagnosis of this form of disease in the United Kingdom.ARTICLE REFERENCES:1. Becker TM, Blount JM, Guinan ME. "Genital herpes infection in private practice in the United States, 1966-1981" . JAMA. 1985; 253:1601-3. Google Scholar2. Lavery HA, Connolly JH, Russell JD. "Incidence of herpes genitalis in N. Ireland in 1973-85 and HSV-1 and 2 isolated in 1982-4" . Genitourinary Med. 1986; 62:24-7. Google Scholar3. Nahmias AJ, Dowdle W. "Antigenic and biologic differences in herpes virus hominis" . 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Google Scholar Previous article Next article FiguresReferencesRelatedDetails Volume 10, Issue 2March 1990 Metrics History Accepted4 June 1989Published online1 March 1990 ACKNOWLEDGMENTWe thank Basrah Univesity for financial support and for granting a 3 months’ sabbatical to Dr. Al-Samarai to allow for research on the topic.InformationCopyright © 1990, Annals of Saudi MedicinePDF download