Comparison of several ELISA tests for detecting the presence of IgG and IgM against herpes simplex viruses

Four enzyme-linked immunosorbent assays designated test 1 (ETI-HSVK-G 1/2); test 2 (ETI-HSVK-M 1/2); test 3 (ETI-HSVK-G 2), and test 4 (BioElisa HSV2 IgG) were studied to evaluate different stages of herpes simplex virus (HSV) infection. Samples (50 sera and 14 cerebrospinal fluid) were included in four groups. Group 1 consisted of samples from patients with primary HSV infections; group 2 comprised samples from patients with recurrent HSV infections; group 3 were samples nonreactive to HSV; and group 4 were samples from patients with infections by other herpes viruses (4a, chickenpox; 4b, herpes zoster; and 4c, infectious mononucleosis by Epstein-Barr virus). The percentages of agreement between tests 1 and 2 were 100 and 72.1%, respectively. The total diagnostic values of tests 1 and 2 were: 100 and 50% sensitivity, respectively; and 100 and 89% specificity, respectively. Few positive results for HSV-2 infection were found, and so, tests 3 and 4 were not evaluated. The results of tests 3 and 4 for a chickenpox patient, and a herpes zoster patient were not in agreement.     

Direct Immunofluorescence Test for the Diagnosis of Genital Herpesvirus Infections

Herpetic lesions of the genitalia may be confused clinically with other ulcerative, genital lesions. Direct immunofluorescence (FA) provides a rapid method of diagnosis, and the utility of this method for the diagnosis of genital ulcers was examined. One hundred and ten patients with genital lesions were examined by darkfield for syphilis and by FA and culture for herpes simplex virus (HSV) infections. Satisfactory samples were obtained from 102 patients, of which 81 were clinically suspected cases of HSV. Acetone-fixed slides of scrapings of ulcerative lesions were stained with conjugated antiserum prepared in rabbits against HSV type 2. HSV was isolated from 73% of specimens of suspected herpetic lesions, and 77% of these specimens were positive by FA. Nine percent were positive by FA only and these were not thought to represent false positives. Five percent were positive by culture only. A comparison of clinical diagnoses with laboratory findings revealed that 4% of the cases were misdiagnosed when only the clinical evaluation was considered. The data suggest that the inclusion of a diagnostic FA test for HSV along with the darkfield examination may be useful for differentiating the etiological agents of ulcerative, genital lesions.     

Herpes Zoster Risk Reduction through Exposure to Chickenpox Patients: A Systematic Multidisciplinary Review

Varicella-zoster virus (VZV) causes chickenpox and may subsequently reactivate to cause herpes zoster later in life. The exogenous boosting hypothesis states that re-exposure to circulating VZV can inhibit VZV reactivation and consequently also herpes zoster in VZV-immune individuals. Using this hypothesis, mathematical models predicted widespread chickenpox vaccination to increase herpes zoster incidence over more than 30 years. Some countries have postponed universal chickenpox vaccination, at least partially based on this prediction. After a systematic search and selection procedure, we analyzed different types of exogenous boosting studies. We graded 13 observational studies on herpes zoster incidence after widespread chickenpox vaccination, 4 longitudinal studies on VZV immunity after re-exposure, 9 epidemiological risk factor studies, 7 mathematical modeling studies as well as 7 other studies. We conclude that exogenous boosting exists, although not for all persons, nor in all situations. Its magnitude is yet to be determined adequately in any study field.     

Analysis of immune function in herpes zoster patients: demonstration and characterization of suppressor cells

We sought to identify imbalances of immune regulatory cells that might contribute to the depression of cell-mediated immunity that occurs during an episode of herpes zoster. Peripheral blood mononuclear cells (PBMC) were obtained from patients with herpes zoster during the acute (less than 7 days after disease onset) and convalescent (more than 10 days after disease onset) phases of illness and from healthy seropositive donors. The PBMC were analyzed for: lymphoproliferative responses to varicella-zoster virus (VZV) antigens, Leu-3 (helper/inducer):Leu-2 (cytotoxic/suppressor) ratios, and percentages of suppressor cells as defined by coexpression of the Leu-2 and OKM1 antigens. Significantly depressed proliferative responses of VZV antigens and Leu-3:Leu-2 ratios, and increased percentages of Leu-2+ OKM1+ suppressor cells were observed in PBMC of acute phase herpes zoster patients as compared with the PBMC of convalescent patients or healthy donors. These differences were also observed in individual patients sequentially studied during both phases of disease. Cryopreserved acute phase PBMC suppressed the proliferative response of autologous convalescent phase PBMC to VZV antigens, but not to herpes simplex virus (HSV) antigens. The acute phase PBMC suppressor cell was radiation sensitive and was identified as a Leu-2+ cell by fluorescence-activated cell sorting. Thus, depression of cell-mediated immunity during the acute phase of herpes zoster was associated with a relative increase of lymphocytes expressing a suppressor cell phenotype and the activation of a radiosensitive Leu-2+ suppressor cell with some degree of antigen specificity.     

Cross-reactivity between herpes simplex virus glycoprotein B and a 63,000-dalton varicella-zoster virus envelope glycoprotein.

Cross-reactive monoclonal antibodies recognizing both herpes simplex virus (HSV) glycoprotein B and a major 63,000-dalton varicella-zoster virus (VZV) envelope glycoprotein were isolated and found to neutralize VZV infection in vitro. None of the other VZV glycoproteins was recognized by any polyclonal anti-HSV serum tested. These results demonstrate that HSV glycoprotein B and the 63,000-dalton VZV glycoprotein share antigenic epitopes and raise the possibility that these two proteins have a similar function in infection.     

Rapid Identification and Typing of Herpes Simplex Virus Types 1 and 2 by a Direct Immunofluorescence Technique

An immunofluorescence (FA) technique has been developed which can identify herpes simplex virus (HSV) in clinical specimens and also type the virus directly as type 1 or type 2. This test, first applied to cervicovaginal specimens obtained from 80 mice genitally inoculated with HSV, indicated a sensitivity approaching 80% in comparison to standard viral isolation methods. A similar sensitivity was found when the test was applied to 185 clinical specimens with adequate cells for staining, which were obtained from a variety of sites of patients with suspect herpetic infection. In only 1 of 6 specimens positive by both FA and culture methods was the HSV type wrongly identified by the FA technique. There were also six specimens which were negative by culture methods but positive by the FA test, indicating a specificity of 91%. It is likely that these are not instances of false-positive tests but of other factors which may have resulted in negative viral isolations by culture methods. As more specific reagents become available, it is anticipated that the FA technique will have wider usage in diagnostic laboratories for the identification and typing of HSV types 1 and 2.     

Varicella-zoster virus (VZV)

Varicella-zoster virus (VZV) causes varicella in primary infection and zoster after reactivation from latency. Both herpes simplex virus (HSV) and VZV are classified into the same alpha-herpesvirus subfamily. Although most VZV genes have their HSV homologs, VZV has many unique biological characteristics. In this review, we summarized recent studies on 1) animal models for VZV infection and outcomes from studies using the models, including 2) viral dissemination processes from respiratory mucosa, T cells, to skin, 3) cellular receptors for VZV entry, 4) functions of viral genes required uniquely for in vivo growth and for establishment of latency, 5) host immune responses and viral immune evasion mechanisms, and 6) varicella vaccine and anti-VZV drugs.     

FTIR spectroscopic method for detection of cells infected with herpes viruses

Microscopic FTIR spectroscopy was used to investigate the spectral differences between normal cells in culture and cells infected with various members of the herpes family of viruses [Herpes simplex (HSV) and Varicella zoster (VZV)]. The main objective of this study is to evaluate the possibility of developing microscopic FTIR spectroscopy as a sensitive assay for the detection of herpetic infections at their early stages. The advantage of this method over conventional FTIR spectroscopy is that it facilitates inspection of restricted regions of tissue. Our results showed significant and consistent differences between all normal and HSV or VZV infected cells that were tested. Detectable and significant spectral differences between normal and infected cells are seen as early as 24 h postinfection, but the damage of the cells (cytopathic effect), caused by the infecting virus, can be seen by optical microscope observations at only 3 days postinfection. An impressive increase in the levels of vital cellular metabolites was seen in the herpes virus infected cells compared to normal cells. It seems that this spectral behavior is unique for infection with herpes viruses, because when these cells were infected with other viruses from different families like retroviruses, a considerable decrease in the levels of vital cellular metabolites was seen in infected cells compared to normal cells. Cluster analysis performed on FTIR mass chromatography yielded 100% accuracy in classifying control uninfected and VZV or HSV infected cells. Our data strongly support the possibility of developing FTIR microscopy as a diagnostic method for early detection of herpetic infections.     

水痘-带状疱疹病毒分离和鉴定

本文报告了用7份水痘或带状疱疹病人的皮肤病变水泡液,在人胚肺二倍体细胞(2BS)中培养传代,分离到7株病毒分离物,第一株是由带状疱疹病例分离到的,经细胞病变、核内包涵体和电镜形态学观察,认为此分离物属于疱疹病毒科成员;经血清学鉴定,证实为水痘-带状疱疹病毒(VZV),其宿主范围与疱疹病毒(HSV)不同,余6株分离物经ELISA、IFA及CF血清学试验鉴定均为VZV。     

Herpesvirus DNA in Peripheral Blood Mononuclear Cells of Some Patients with Meniere's Disease

Herpes simplex virus‐1 (HSV) or varicella zoster virus (VZV) DNA was detected by nested polymerase chain reaction in peripheral blood mononuclear cells of patients with Meniere's disease (one of 28 patients for HSV‐1,2 of 28 patients for VZV) during acute illness (within 5 days after onset). On the other hand, neither HSV‐1 DNA or VZV DNA was detected in PBMCs of 50 age‐ and sex‐matched healthy individuals and 50 pregnant women. These findings may imply that reactivation of HSV‐1 or VZV may be associated with the development of some cases of Meniere's disease.     

Comparison of real-time and nested PCR assays for detection of herpes simplex virus DNA

We performed a real-time PCR assay to detect herpes simplex virus (HSV) DNA, and compared it prospectively with a nested PCR assay in 164 clinical samples (109 cerebrospinal fluid and 55 sera) from patients suspected of having neonatal HSV infection or HSV encephalitis. In 25 of 164 samples, HSV DNA was detected by the nested PCR assay. All samples positive for HSV DNA in the nested PCR assay were also positive in the real-time PCR assay, and all but two samples negative for HSV DNA in the nested assay were negative in the real-time assay. The real-time PCR assay thus had a sensitivity of 100% and a specificity of 99%, when compared with the nested assay. Sequential assays in a case of disseminated HSV showed that a decrease in HSV DNA paralleled clinical improvement. Quantification of HSV DNA by real-time PCR was useful for diagnosing and monitoring patients with HSV encephalitis and neonatal HSV infection.     

Etiologic Agents of Central Nervous System Infections among Febrile Hospitalized Patients in the Country of Georgia

Objectives

There is a large spectrum of viral, bacterial, fungal, and prion pathogens that cause central nervous system (CNS) infections. As such, identification of the etiological agent requires multiple laboratory tests and accurate diagnosis requires clinical and epidemiological information. This hospital-based study aimed to determine the main causes of acute meningitis and encephalitis and enhance laboratory capacity for CNS infection diagnosis.

Methods

Children and adults patients clinically diagnosed with meningitis or encephalitis were enrolled at four reference health centers. Cerebrospinal fluid (CSF) was collected for bacterial culture, and in-house and multiplex RT-PCR testing was conducted for herpes simplex virus (HSV) types 1 and 2, mumps virus, enterovirus, varicella zoster virus (VZV), Streptococcus pneumoniae, HiB and Neisseria meningitidis.

Results

Out of 140 enrolled patients, the mean age was 23.9 years, and 58% were children. Bacterial or viral etiologies were determined in 51% of patients. Five Streptococcus pneumoniae cultures were isolated from CSF. Based on in-house PCR analysis, 25 patients were positive for S. pneumoniae, 6 for N. meningitidis, and 1 for H. influenzae. Viral multiplex PCR identified infections with enterovirus (n = 26), VZV (n = 4), and HSV-1 (n = 2). No patient was positive for mumps or HSV-2.

Conclusions

Study findings indicate that S. pneumoniae and enteroviruses are the main etiologies in this patient cohort. The utility of molecular diagnostics for pathogen identification combined with the knowledge provided by the investigation may improve health outcomes of CNS infection cases in Georgia.     

Passive Hemagglutination Assays for the Detection of Antibodies to Herpes Viruses

A simple and effective method for the detection of antibodies to herpes simplex virus (HSV), human cytomegalovirus (HCMV) and varicella-zoster virus (VZV), has been established using the passive hemagglutination assay (PHA) in combination with viral specific glycoproteins. The results obtained with the PHA were compared with those from neutralization (NT) and complement fixation (CF) tests. The PHA test for each of the herpes viruses appears to compare favorably with the other assays tested. The specificity and sensitivity of HSV PHA to NT were 100%, whereas the specificity and sensitivity of HSV CF test to NT were 98% and 100%, respectively. For HCMV, the specificity and sensitivity of PHA to NT and PHA to CF were 100%. Similarly, the specificity and sensitivity of VZV PHA to NT were 100%. Because of the low sensitivity of the VZV CF, the sensitivity of CF to NT was 83%. Furthermore, the range of antibody titers and their absolute levels obtained in the PHAs were significantly greater than those in the NT and CF tests.     

Utilization of herpes simplex PCR assays for cerebrospinal fluid in a pediatric health care setting

An assessment was made of the utilization and impact of a diagnostic polymerase chain reaction (PCR) assay for the diagnosis of herpes simplex viruses (HSV) 1 and 2 in cerebrospinal fluid of children who attended a Canadian pediatric referral centre. One hundred and three assays were performed on specimens from 103 patients during the period August 1997 to September 1998. Patient ages ranged from newborn to 16 years. Indications for HSV PCR included seizures with or without fever (56.3%), aseptic meningitis (16.5%), and encephalopathy with or without fever (10.7%). Only 2 of 103 (1.9%) assays were positive, including one each for HSV1 and HSV2. Control specimens that were seeded with virus indicated inhibition for 24.3, 8.8, and 6.8% of assays for HSV1, HSV2, and both HSV1 and HSV2, respectively. The mean turn-around time for HSV PCR was 2.5 days, and 90.3% were completed in less than 5 days. Acyclovir was administered to 78.6% of the patients overall; the results of the HSV PCR impacted on the treatment courses for 36 individuals. Nevertheless, 16.5% of patients continued to receive extended courses of antiviral therapy despite negative HSV PCR assays. Although it is desirable to decrease the frequency of PCR inhibitions and to further decrease the interval to assay completion, HSV PCR does have a significant impact on antiviral use in this setting.     

Evaluation of Herpes Simplex Detection in Corneal Scrapings by Three Molecular Methods

Herpes simplex virus (HSV)-1 keratitis (HSK) is a sight-threatening ocular infection with worldwide occurrence. A prompt laboratory diagnosis is often very useful. The purpose of this study was to evaluate molecular methods as rapid diagnostic tools compared with cell culture of HSK. Corneal scrapings from patients with clinically suspected HSK were tested by direct immunofluorescence assay (IFA) for HSV-1 antigen and by polymerase chain reaction (PCR) for HSV-1 DNA, and an attempt for viral isolation was performed on Vero cell line culture. Positive samples by cell culture were 20.8%, whereas PCR was positive in 29.2%, and IFA was positive in 33.3%. IFA had better sensitivity (80%) and negative predictive value (81.8%) than PCR (70% and 76.9%, respectively); however, PCR had better specificity (71.4%) and positive predictive value (63.6%). This indicates that a combination of cell culture, IFA and PCR constitutes the best set of tools for diagnosis of clinically suspected cases of HSK. Documented infection can be further assessed by cell-culture technique or PCR depending laboratory availability.     

Characterization of Varicella-Zoster virus glycoprotein K (open reading frame 5) and its role in virus growth

Varicella-zoster virus (VZV) is an alphaherpesvirus that is the causative agent of chickenpox and herpes zoster. VZV open reading frame 5 (ORF5) encodes glycoprotein K (gK), which is conserved among alphaherpesviruses. While VZV gK has not been characterized, and its role in viral replication is unknown, homologs of VZV gK in herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) have been well studied. To identify the VZV ORF5 gene product, we raised a polyclonal antibody against a fusion protein of ORF5 codons 25 to 122 with glutathione S-transferase and used it to study the protein in infected cells. A 40,000-molecular-weight protein was detected in cell-free virus by Western blotting. In immunogold electron microscopic studies, VZV gK was in enveloped virions and was evenly distributed in the cytoplasm in infected cells. To determine the function of VZV gK in virus growth, a series of gK deletion mutants were constructed with VZV cosmid DNA derived from the Oka strain. Full and partial deletions in gK prevented viral replication when the gK mutant cosmids were transfected into melanoma cells. Insertion of the HSV-1 (KOS) gK gene into the endogenous VZV gK site did not compensate for the deletion of VZV gK. The replacement of VZV gK at a nonnative AvrII site in the VZV genome restored the phenotypic characteristics of intact recombinant Oka (rOka) virus. Moreover, gK complementing cells transfected with a full gK deletion mutant exhibited viral plaques indistinguishable from those of rOka. Our results are consistent with the studies of gK proteins of HSV-1 and PRV showing that gK is indispensable for viral replication.     

Genome-Wide Analysis of T Cell Responses during Acute and Latent Simian Varicella Virus Infections in Rhesus Macaques

Varicella zoster virus (VZV) is the etiological agent of varicella (chickenpox) and herpes zoster (HZ [shingles]). Clinical observations suggest that VZV-specific T cell immunity plays a more critical role than humoral immunity in the prevention of VZV reactivation and development of herpes zoster. Although numerous studies have characterized T cell responses directed against select VZV open reading frames (ORFs), a comprehensive analysis of the T cell response to the entire VZV genome has not yet been conducted. We have recently shown that intrabronchial inoculation of young rhesus macaques with simian varicella virus (SVV), a homolog of VZV, recapitulates the hallmarks of acute and latent VZV infection in humans. In this study, we characterized the specificity of T cell responses during acute and latent SVV infection. Animals generated a robust and broad T cell response directed against both structural and nonstructural viral proteins during acute infection in bronchoalveolar lavage (BAL) fluid and peripheral blood. During latency, T cell responses were detected only in the BAL fluid and were lower and more restricted than those observed during acute infection. Interestingly, we identified a small set of ORFs that were immunogenic during both acute and latent infection in the BAL fluid. Given the close genome relatedness of SVV and VZV, our studies highlight immunogenic ORFs that may be further investigated as potential components of novel VZV vaccines that specifically boost T cell immunity.     

Diagnosis of herpes simplex virus in routine smears by an immunoperoxidase technique

Routine Papanicolaou-stained cervicovaginal smears from 59 patients were cytologically screened for herpetic infection. Forty-one of the smears were positive for herpes, 2 were suspicious and 16 were negative. All 59 slides were then destained and restained by a commercial immunoperoxidase kit for the detection of herpes simplex virus (HSV). The immunoperoxidase stain was positive in 23 of the 41 cytologically positive slides. One of the 2 cytologically suspicious slides was also immunoperoxidase positive, as was 1 of the 16 cytologically negative slides. This study indicates that immunoperoxidase staining is very specific but not quite as sensitive as routine Papanicolaou-stained smears in the detection of HSV. The immunoperoxidase method is thus recommended for the confirmation of HSV cases rather than for the routine diagnosis of HSV infection.