Comparison of viral isolation, direct immunofluorescence, and indirect immunoperoxidase techniques for detection of genital herpes simplex virus infection.

Seventy-six consecutive patients presenting to a genital herpes simplex virus (HSV) clinic were enrolled in a study comparing viral isolation (VI), indirect immunoperoxidase (indirect IP), and direct immunofluorescence (direct FA) techniques for the detection of HSV antigen. Of the 76 patients, 61 (80%) demonstrated HSV by VI, compared with 66% by indirect IP and 55% by direct FA (P less than 0.05). Genital lesions from nine patients demonstrated HSV antigen by direct FA or indirect IP but were VI negative; eight of nine patients had subsequent episodes of genital HSV confirmed by VI. During the vesicular-pustular stage of the disease, VI was positive in 90%, indirect IP was positive in 76%, and direct FA was positive in 71% of the lesions, whereas with ulcerative lesions, VI was positive in 72%, indirect IP was positive in 55%, and direct FA was positive in 38%. These commercially available rapid viral diagnostic techniques are specific and useful, if adequate specimens are obtained from early genital lesions.     

Recurrences after oral and genital herpes simplex virus infection. Influence of site of infection and viral type

We prospectively followed 39 adults with concurrent primary herpes simplex virus (HSV) infection (12 with HSV type 1 and 27 with HSV type 2) of the oropharynx and genitalia, caused by the same virus in each person, to evaluate the influence of viral type (HSV-1 vs. HSV-2) and site of infection (oropharyngeal vs. genital) on the frequency of recurrence. The subsequent recurrence patterns of HSV infection differed markedly according to viral type and anatomical site. Oral-labial recurrences developed in 5 of 12 patients with HSV-1 and 1 of 27 patients with HSV-2 (P less than 0.001). Conversely, genital recurrences developed in 24 of 27 patients with HSV-2 and 3 of 12 patients with HSV-1 (P less than 0.01). The mean rate of subsequent genital recurrences (due to HSV-1 and HSV-2) was 0.23 per month, whereas the mean rate of oral-labial recurrences was only 0.04 per month (P less than 0.001). The mean monthly frequencies of recurrence were, in order, genital HSV-2 infections, 0.33 per month; oral-labial HSV-1 infections, 0.12 per month; genital HSV-1 infections, 0.020 per month; and oral HSV-2 infections, 0.001 per month (P less than 0.01 for each comparison). We conclude that the likelihood of reactivation of HSV infection differs between HSV-1 and HSV-2 infections and between the sacral and trigeminal anatomical sites. The sixfold more frequent clinical recurrence rate of genital HSV infections as compared with oral-labial HSV infections may account for the relatively rapid increase in the prevalence of clinically recognized genital herpes in recent years.     

Cellular immune response in genital herpes simplex virus infection.

We studied the relations between the cellular immune response, pre-existing complement-fixing antibody and virus type with duration of virus excretion in genital herpes simplex virus (HSV) infection. Thirty-six patients (seven with HSV-1 and 29 with HSV-2) with genital herpes underwent serologic testing, sequential viral cultures and weekly determination of lymphocyte-transformation stimulation index with inactivated HSV antic n. The duration of virus excretion was shortest in those with pre-existing complement-fixing antibody, was unrelated to virus type, and was inversely correlated with the magnitude of the mean peak stimulation index (r = -0.69, P less than 0.001). Prolonged virus excretion occurred in patients with a delayed and diminished peak index. Recurrent episodes had a higher peak index (29.4 compared to 14.5) (P less than 0.02), an earlier development of the peak during recurrences (9.1 vs. 25.8 days) (P less than 0.01) and a briefer duration of viral shedding than initial episodes. Thus, the temporal course and magnitude of the stimulation index correlate with and may determine the duration of genital HSV infection.     

Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections

A novel multiplex nested polymerase chain reaction (PCR) assay was designed and evaluated for routine diagnosis of herpes simplex virus (HSV) infections in patients with either putative HSV infection of the central nervous system or suspected HSV keratitis. Single-tube amplification of HSV type 1 (HSV-1) or type 2 (HSV-2) DNA extracted from cerebrospinal fluid (CSF) or from keratectomy specimens was followed by differentiation of the virus type-specific PCR products either by agarose gel analysis or by DNA enzyme immunoassay. Among 417 CSF specimens obtained from 395 consecutive patients with clinically suspected HSV infection, 11 (2.6%) were positive for HSV-1 DNA and four (1.0%) probes were positive for HSV-2 DNA. None of the specimens was positive for both HSV-1 and HSV-2 DNA. The genome of HSV-2 was detected in a CSF sample obtained from a woman with meningoencephalitis and genital herpes. The presence of PCR inhibitors was detected in six of 111 (5.4%) reconstructed CSF samples. Inhibition could be removed following extraction with a commercial kit. HSV-1 DNA, but no HSV-2 DNA, was detected in corneal buttons obtained from patients with suspected herpetic keratitis. No contamination has been recorded during the 2-year routine use of this test, which has met the specific requirements of a diagnostic laboratory. © 1996 Wiley-Liss, Inc.     

Evaluation of biotin-streptavidin enzyme-linked immunosorbent assay for detection of genital herpes simplex virus infection

A biotin-streptavidin enzyme-linked immunosorbent assay (B-SA ELISA) was evaluated for detection of herpes simplex virus (HSV) in clinical specimens which were cervico-vaginal swabs from 205 asymptomatic women and swabs from the genital lesions of 163 suspected patients. All specimens were also subjected to a conventional virus isolation in cell culture. A blocking B-SA ELISA had 100% specificity and 98% sensitivity compared with viral isolation from patients, but had only 40% sensitivity using specimens from asymptomatics. The conventional B-SA ELISA might also be used; it gave results corresponding to B-SA ELISA blocking test except for a single specimen which was considered a false positive.     

Evaluation of a quantitative competitive PCR assay for measuring herpes simplex virus DNA content in genital tract secretions.

Previous studies have shown an association between the approximate titer of herpes simplex virus (HSV) DNA in clinical specimens and the ability to isolate HSV from genital secretions. To control for variance in amplification conditions, we developed a competitive quantitative PCR (QC PCR) for the detection of HSV DNA. The assay accurately measured from 10 to 10(6) copies of HSV DNA. We compared the QC PCR with our previous semiquantitative detection method and found concordance for 61 of 63 positive specimens. We also evaluated the HSV DNA content from individual swabs of genital secretions obtained from individual sites of the genital tract (cervix, vulva, and rectum) with that from one swab with secretions from all three sites. The concordance for detecting HSV DNA was 91%; for only 4 of 143 collection days was there a > 1 log difference between the two collection methods. A single swab with secretions from all three genital sites and evaluated in a QC PCR format can accurately measure the frequency of subclinical and clinical shedding of HSV and the titer of HSV shed from the genital region. Such an approach should be very useful in the evaluation of antiviral chemotherapy for HSV.     

Pathogenesis of genital herpes simplex virus infection in mice

This study was undertaken to establish the role of virulence of various herpes simplex virus (HSV) strains in the course of infection when applying the virus to the non-injured mucous membranes of mice.Wild-type HSV-type 1 (HSV-1) strains with marked differences in their neurovirulence following intracerebral inoculation showed minor differences in virulence after vaginal inoculation, but essentially their neurovirulence in cerebral infection corresponded to their virulence on the mucous membranes.In comparison with the wild-types, however, there were pronounced differences among syn- and TK^–-mutants of HSV-1 and HSV-2 in the degree of virulence at different sites in the course of virus infection. Whereas syn-mutants proved avirulent on the mucous membranes but not in neural tissues, TK^–-mutants were avirulent both on mucous membranes and in neural tissues.Ts-mutants of HSV-2 were not found to establish themselves when administered to the non-injured mucous membranes, nor did they induce neutralizing antibodies, but a later challenge with the wild-type virus at the same site lead only to an attenuated course of infection.Supported by the Deutsche Forschungsgemeinschaft Schn 174/6-2     

Pathogenesis of genital herpes simplex virus infection in mice

Experiments in the mouse model of herpes simplex virus (HSV) infection involving the intact genital mucous membranes as inoculation site yielded the following results. In untreated mice the extent of latency was correlated with the degree of peripheral virus replication. This correlation could not be observed when the course of infection was interrupted by chemotherapy, interferon, or passive immunization. Acyclovir had little effect on peripheral virus multiplication, but markedly reduced latent ganglionic infection. As acute ganglionic infection and virus concentration in the spinal nerves were already reduced, acyclovir is assumed to inhibit either virus penetration into the nerve endings or virus replication in the ganglia. Interferon apparently has an active role in the elimination of virus infected cells from the ganglia, as its effect was restricted to a reduced rate of latency and of lethality. Passive immunization with antiserum led to similar results as ACV-treatment. While lacking a pronounced effect on virus replication in the mucous membranes, specific antibody was found to influence both virus elimination from the ganglia, and conversion from productive to latent ganglionic infection. Immune lymphocytes proved to be the only agent capable of suppressing peripheral infection, thereby inhibiting the neural spread of the virus. These results suggest that the decrease in latency may result from modulations occurring at different stages in the course of infection.Supported by Deutsche Forschungsgemeinschaft Schn 174/6-2     

Inapparent genital herpes simplex virus infection in college women

During a six-month period, 600 gynecological samples were collected from 585 women with typical herpes lesions, women with non-herpes symptoms (ie, vaginitis, moniliasis, trichomoniasis, etc), and normal women seen at the student health center gynecological clinic and processed for herpes simplex virus (HSV) isolation. From these specimens, 29 samples from 25 of the 585 women (4.3%) were positive for HSV. When these isolates were typed using plaque diameter in chick cells, heat stability of viral thymidine kinase (T.K.), and restriction endonuclease patterns it was found that 18 samples (15 patients or 60%) were HSV-2 and 11 samples (10 patients or 40%) were HSV-1. Inapparent HSV infections constituted 20.0% of the virologically confirmed samples (5 of 25 patients) and represented 0.9% of the total patients studied (5 of 585). The inapparent infections were about equally divided between the two HSV types (2 were HSV-2 and 3 were HSV-1), and 4 of 5 occurred in the presence of clinically diagnosed monilia.     

Diagnosis of herpes simplex virus by direct immunofluorescence and viral isolation from samples of external genital lesions in a high-prevalence population.

One hundred thirty specimens from 108 consecutive patients with a history of recurrent genital ulcerations were used in a study comparing herpes simplex virus (HSV) isolation with a direct fluorescent-antibody (DFA) technique using mouse monoclonal antibodies. HSV was isolated from 70% of vesicular lesions, 67% of pustular lesions, 32% of ulcerative lesions, and 17% of crusted lesions, whereas the DFA technique detected HSV antigen in 87, 67, 30, and 10% of lesions in similar stages, respectively. When both methods were used, HSV was identified in 97, 79, 45, and 17% of vesicles, pustules, ulcers, and crusted lesions, respectively. The overall sensitivity and specificity of the DFA technique in comparison with virus isolation (VI) were 74 and 85%, respectively. Of the 17 patients from whom DFA-positive, VI-negative samples were obtained, HSV was subsequently isolated from a genital lesion in 14, suggesting that they were not DFA false-positives. Similarly, of the 46 patients whose initial lesion samples were DFA and VI negative, 37 (80%) had HSV identified from subsequent genital lesions during follow-up. Thus, a single sample for VI or DFA testing from a recurrent genital lesion had a sensitivity of only 53 and 51%, respectively. Combining the DFA technique and VI increased the sensitivity of laboratory diagnosis of a single recurrent episode of genital HSV; however, repetitive laboratory testing was often required to confirm the diagnosis of recurrent genital HSV infection.     

Epidemiology of genital herpes simplex virus infection in developed countries

Comparisons of the seroepidemiology of genital herpes simplex virus (HSV) infection within and between countries are hampered by variations in tests, methods and populations sampled. Differences in seroprevalence may partly reflect variability in diagnostic efforts and healthcare awareness, expectations and utilization. To allow comparison between surveys and to improve their performance, seroepidemiological studies should use validated HSV type-specific tests, report age-specific or age-adjusted prevalence and define the period of time over which samples were collected. Despite the difficulty of comparing studies, the prevalence of HSV-2 infection varies between developed countries. Among healthy adult populations, HSV-2 seroprevalence is higher in the USA than in Europe. Furthermore, HSV-2 seroprevalence varies widely among European countries. For example, in 1989 HSV-2 seroprevalence among pregnant women was reported to be 33% in Sweden compared with 8.3% in Germany. In some, but not all, countries, HSV-2 seroprevalence appears to be increasing. In the USA, the National Health and Nutrition Examination Surveys found that HSV-2 seroprevalence increased by almost one third from 16.4% to 21.8% from 1976 to 1994 in people over 12 years old. The incidence of HSV infection is a measure of primary infection. HSV incidence is difficult to quantify, partly due to unrecognized or asymptomatic infections. However, estimates of incidence in North American and European populations range from 5 to 24 per 100 people per year. Prevention programmes should recognize that HSV-2 seroprevalence increases rapidly in early adult life. The proportion of genital herpes infections caused by HSV-1 is increasing in the developed world, possibly due to changes in oral-genital sexual behaviour and lower rates of HSV-1 acquisition in childhood.     

Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation.

BACKGROUND: Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) cause a wide range of signs and symptoms, varying from trivial mucocutaneous lesions to life-threatening infections, especially in immuno-suppressed patients. Since antiviral drugs are available, rapid and sensitive laboratory diagnosis of these virus infections is important. OBJECTIVE: To set up and evaluate HSV-1, HSV-2 and VZV qualitative real-time PCR on the Lightcycler system and to compare the results with those of the 'in-house' nested PCR and virus isolation. STUDY DESIGN: 110 consecutive samples from dermal or genital lesions from patients suspected of having HSV infections and another 110 samples from patients with suspected VZV infections were tested with real-time PCR, nested PCR and virus isolation. RESULTS: 24 samples (22%) were positive for HSV-1 by virus isolation and nested PCR, whereas 26 (24%) were positive by real-time PCR. HSV-2 was detected in 28 samples (25%) by virus isolation, in 41 (37%) by nested PCR and in 40 (36%) by real-time PCR. VZV was isolated in 15 samples (14%) and VZV DNA was detected in 51 samples (46%) by nested PCR as well as by real-time PCR. Nucleic acid amplification increased the detection rate of HSV-2 and VZV DNA in particular compared to virus isolation. No significant difference in sensitivity was found between real-time PCR and nested PCR. CONCLUSION: Real-time PCR has the advantage of rapid amplification, a reduced risk for contamination and it is a suitable method for diagnosis of VZV and HSV in specimens from skin lesions.     

Typing of herpes simplex virus isolates by enzyme-linked immunosorbent assay: comparison between indirect and double-antibody sandwich techniques.

Indirect and double-antibody sandwich enzyme-linked immunosorbent assay techniques were compared for serotyping 42 herpes simplex virus isolates. Both procedures gave similar results, which were in complete agreement with those obtained by inhibition of the indirect hemagglutination test. The indirect technique proved to be as specific as the double-antibody sandwich enzyme-linked immunosorbent assay, but simpler and cheaper.     

Virus-specific antibodies in sera from patients with genital herpes simplex virus infection.

Virus-specific antibodies against a number of herpes simplex virus type 2 antigens were determined by radioimmunoprecipitation assays in sequential serum samples obtained from 12 patients with initial genital herpes simplex virus infection. The progressive appearance of antibodies to virus-specific antigens was observed; antibodies against a 130,000-molecular-weight glycoprotein complex appeared first, followed by antibodies against the major nucleocapsid polypeptide and then antibodies against a number of other viral antigens, including a polypeptide with a molecular weight of 62,000. Patients who developed a wide variety of antibodies to viral polypeptides shortly after resolution of their initial episode seemed to experience more severe initial infections and more recurrences than did those who reacted poorly with these virus-specific antigens. This was most apparent with respect to antibodies to virus-specific polypeptides with molecular weights between 30,000 and 43,000. Antibody specificity did not change during the course of follow-up regardless of whether serum samples were taken shortly before, during, or after recurrent episodes. Glycoprotein-specific antibodies were quantitated with the purified 130,000-molecular-weight glycoprotein material. No significant fluctuations in these antibody titers were observed before or after recurrences of the disease.     

A comparison of PCR with virus isolation and direct antigen detection for diagnosis and typing of genital herpes

Patients attending the genitourinary medicine clinic at Watford General Hospital, UK, were examined for clinical signs of genital herpes infection. Genital swabs were taken from 194 patients (126 female, 68 male) who presented with genital ulceration or symptoms which were suggestive of genital herpes infection. Swabs from these patients were tested by three methods: (i) Detection of herpes simplex virus (HSV) antigen by direct HSV enzyme immunoassay (EIA), (ii) HSV isolation in Vero cell culture and (iii) HSV polymerase chain reaction (PCR). HSV was detected in 76 patients (39%) by EIA, in 93 (48%) by isolation in cell culture, and in 115 (59%) by PCR. Isolation by cell culture has been considered as the “gold standard” for the detection of HSV in genital lesions, but in this study HSV PCR was significantly more sensitive. Comparison of the three methods was as follows: Cell culture vs. PCR: Sensitivity 93/115 (80.9%), Specificity 79/79 (100%). HSV EIA vs. PCR: Sensitivity 75/115 (65.2%), Specificity 78/79 (98.7%). HSV EIA vs. Cell culture: Sensitivity 75/93 (80.7%), Specificity 100/101 (99%). EIA was less effective in detecting HSV among recurrent than among first episode infections, in comparison to culture or HSV PCR. This is the first comparison of HSV PCR with two other routine diagnostic methods for confirming genital herpes infection in a symptomatic population. The infecting HSV type was identified by restriction digestion of 108 HSV amplicons: HSV-1: 37/108 (34%), HSV-2: 71/108 (66%). In this population HSV-1 causes a significant proportion of genital herpes cases, and HSV-1 genital infection was detected in significantly more first episode infections (40.3%) than among recurrent infections (22.2%). J. Med. Virol. 55:177–183, 1998. © 1998 Wiley-Liss, Inc.     

Comparison of loop-mediated isothermal amplification, real-time PCR, and virus isolation for the detection of herpes simplex virus in genital lesions

This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions; two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samples testing positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%.     

Quantitation of viral load in neonatal herpes simplex virus infection and comparison between type 1 and type 2

Neonatal herpes simplex virus (HSV) infection is a severe disease with high mortality and morbidity in spite of the development of effective anti-viral therapies. The viral load in neonatal herpes simplex virus (HSV) infection was measured retrospectively in 37 patients. HSV DNA copy numbers in serum and cerebrospinal fluid (CSF) were quantified using a real-time PCR assay. Patients with disseminated infection had a higher viral load in their sera. whereas patients with central nervous system (CNS) infection exhibited a higher viral load in the CSF. The viral load was significantly higher in the serum of patients who died later. Interestingly, patients with HSV type-2 infection exhibited more CNS involvement and neurological impairment, together with a high viral load in the CSF, than did HSV type-1 patients. These results suggest that quantitation of HSV viral load may be useful for assessing the prognosis, and may provide additional information for the management of neonatal HSV infection.     

Reactivation of genital herpes simplex virus type 2 infection in asymptomatic seropositive persons

BACKGROUND: Most persons who have serologic evidence of infection with herpes simplex virus (HSV) type 2 (HSV-2) are asymptomatic. Historically, it has been assumed that these persons have less frequent viral reactivation than those with symptomatic infection. METHODS: We conducted a prospective study to investigate genital shedding of HSV among 53 subjects who had antibodies to HSV-2 but who reported having no history of genital herpes, and we compared their patterns of viral shedding with those in a similar cohort of 90 subjects with symptomatic HSV-2 infection. Genital secretions of the subjects in both groups were sampled daily and cultured for HSV for a median of 94 days. RESULTS: HSV was isolated from the genital mucosa in 38 of the 53 HSV-2-seropositive subjects (72 percent) who reported no history of genital herpes, and HSV DNA was detected by the polymerase-chain-reaction assay in cultures prepared from genital mucosal swabs in 6 additional subjects. The rate of subclinical shedding of HSV in the subjects with no reported history of genital herpes was similar to that in the subjects with such a history (3.0 percent vs. 2.7 percent). Of the 53 subjects who had no reported history of genital herpes, 33 (62 percent) subsequently reported having typical herpetic lesions; the duration of their recurrences in these subjects was shorter (median, three days vs. five days; P<0.001) and the frequency lower (median, 3.0 per year vs. 8.2 per year; P<0.001) than in the 90 subjects with previously diagnosed symptomatic infection. Only 1 of these 53 subjects had no clinical or virologic evidence of HSV infection. CONCLUSIONS: Seropositivity for HSV-2 is associated with viral shedding in the genital tract, even in subjects with no reported history of genital herpes.     

Virological diagnosis of herpes simplex virus 1 esophagitis by quantitative real-time PCR assay

Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the "gold standard." From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain.