Detection Of Herpes Simplex Virus Antigen Research Articles

Protein A-Based ELISA: Its Evaluation in the Diagnosis of Herpes Simplex Encephalitis

Herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system. As effective antiviral drugs are available, early rapid and reliable diagnosis has become important. The objective of the present study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) protocol for herpes simplex virus (HSV) antigen detection by assessing the usefulness of hyperimmune sera isolated from HSV-seropositive patients. A total of 52 cerebrospinal fluid (CSF) and 62 serum samples of HSE patients and non-HSE persons were analyzed. An in-house ELISA protocol utilizing hyperimmune sera was developed for HSV antigen detection. To improve the specificity of the method, protein A was incorporated into the protocol for ELISA. The sensitivity (70% and 90%) of antigen detection was high in CSF and serum samples, respectively, of confirmed HSE patients. However, lower specificity (52.3% and 42.3%), respectively, was obtained, which was improved by using protein A in the ELISA protocol. The modification in the method yielded good sensitivity (80% and 70%) and specificity (85.7% and 88.4%) of HSV antigen detection in the CSF and sera, respectively, of the HSE and non-HSE groups. The ELISA method utilizing hyperimmune sera along with protein A for HSV antigen detection yielded good sensitivity and specificity in both CSF and sera, and hence can be useful for the diagnosis of HSE.

Influence of Human Immunodeficiency Virus Status on the Clinical History of Herpes simplex Keratitis

Human immunodeficiency virus (HIV) infection is associated with a wide spectrum of systemic and ocular infectious diseases. Little is known about its association with herpes simplex keratitis (HSK) in this geographical region (South India). A retrospective study was undertaken to analyze this association in a cohort of 30 virologically proven recurrent HSK cases. Laboratory methods included herpes simplex virus (HSV) isolation, HSV antigen detection and tear secretory IgA or HSV DNA detection while commercial ELISA kits detected HIV infection. The rationale behind the HIV screening was to assess the role of HIV with increased HSK recurrences. Confirmed HIV seropositivity was 16.7% in recurrent HSK cases as against 3.3% in the matched first-episode HSK cases (p < 0.05, Fisher’s exact test). Our observations on the features of herpetic keratitis in HIV-proven patients, though based on a small number of cases, raise the question whether the immunological abnormalities associated with HIV/AIDS may affect the clinical course of HSK.

Polymerase chain reaction in the diagnosis of herpetic keratitis: experience in a developing country

Background: Herpetic ocular disease is a major cause of blindness. Rapid and accurate diagnosis is essential for prompt, proper treatment. We evaluated the usefulness of detection of herpes simplex virus (HSV) DNA by polymerase chain reaction (PCR) in the laboratory diagnosis of herpetic keratitis.Methods: A retrospective study was conducted involving 234 patients who attended the cornea clinic at the Regional Ophthalmic Institute, Chennai, India, between March 1995 and September 1997. Inclusion in the study was based on clinical diagnosis of herpetic keratitis. Oligonucleotide primers directed against the HSV-I thymidine kinase gene were used, yielding a 110 base pair amplicon. The utility of PCR analysis was assessed against other diagnostic markers: HSV isolation on cell culture, HSV antigen detection by indirect immunofluorescence, detection of antiHSV IgG by enzyme-linked immunosorbent assay (ELISA) and detection of HSVspecific tear secretory IgA (sIgA) by ELISA. These tests showed overall sensitivity values of 22.4%, 39.8%, 30.4% and 20.3% respectively.Results: In epithelial keratitis all 35 specimens from which virus was cultured were positive by PCR. PCR gave a positive result in 23 (82. I %) of the 28 specimens in which HSV antigen was detected and in 4 (57. I %) of the 7 specimens that showed HSV-specific IgG. In addition, PCR detected HSV DNA in 5 of the 30 cases in which these three tests gave a negative result. PCR of two pooled tear samples (collected I week apart from the same patient) from 40 patients with stromal keratitis gave a positive result in 12 cases (30%). In stromal keratitis the sensitivity of PCR in detecting HSV DNA in tear samples was 85.7% with culture, indirect immunofluorescence and detection of anti-HSV IgG as the gold standard, and 80% with detection of sIgA as the gold standard.Interpretation: The results confirm the good correlation with the clinical picture that can be obtained with PCR analysis. They also highlight the diagnostic utility of PCR in detecting HSV DNA in tear samples. This is particularly important in herpetic stromal keratitis, in which collection of corneal scrapings is not advised and, hence, conventional techniques such as virus isolation and antigen detection become difficult.

Herpes Simplex Keratitis in South India: Clinico-Virological Correlation

Purpose: A retrospective cross-section study to analyze the prevalence of herpes simplex virus–induced keratitis (HSK) among 3,000 patients attending a corneal clinic in South India between 1995 and 1997, and to evaluate laboratory techniques for detecting HSK. Methods: The clinico-virological correlation was studied using herpes simplex virus (HSV) isolation on the Vero cell line, HSV-specific antigen detection by indirect immunofluorescence (IF) microscopy, and serum anti-HSV IgG quantitation, IgM estimation, and tear secretory IgA (sIgA) detection by ELISA. Observations: HSK had a prevalence of 7.8% (234 patients) in this study. A virological correlation could be obtained in 44.4% of the cases that had epithelial manifestations and in 14.8% of the cases that had only stromal disease. In 161 cases where both culture and IF microscopy were used, IF detected 27 cases (26.8%) more than cell culture. The difference in sensitivity between cell culture and IF was found to be statistically significant (McNemar's test, P < .05). An elevation in IgG titer was seen in 17 (30.4%) cases. IgM was detected in only 2 cases of the 62 (3.2%) analyzed. Of the 138 cases analyzed, sIgA was positive in 28 (20.3%) cases. A proved diagnosis could be made in 58% of cases when the specimen was collected during the first week after disease onset, and in only 5% when the time interval increased to 4 weeks. Conclusions: HSV antigen detection by indirect IF is a rapid and sensitive diagnostic tool for HSK. Tear secretory IgA (sIgA) is a specific marker for acute herpetic keratitis, and the detection of HSV-specific tear sIgA is a valuable adjunct to virus isolation and antigen detection in the laboratory diagnosis of HSK. For a successful diagnosis, the specimen should be collected as soon as possible after HSK onset.

Tear secretory IgA: evaluation of usefulness as a diagnostic marker in herpetic keratitis

In a South Indian study, an `in-house' enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the potential of herpes simplex virus (HSV)-specific tear secretory IgA (sIgA) in the diagnosis of herpes simplex keratitis (HSK). The presence of HSV-specific tear sIgA was found to be diagnostic in 20.28% of cases. The usefulness of the sIgA ELISA system was evaluated against HSV isolation, which is the `gold standard' and HSV antigen detection, a more sensitive, commonly employed method. Analysis of HSV-specific IgG and IgM results showed their failure as reliable indicators of active or ongoing infection. Comparison of sIgA ELISA with culture as `gold standard' showed its sensitivity, specificity, and positive and negative predictive values to be 60% (95% CI 36.4-80), 93.2% (95% CI 86.7-96.8), 60% (95% CI 36.4-80), and 93.2% (95% CI 86.7-96.8), respectively. This study is the first report on the complete evaluation of the usefulness of tear anti-HSV sIgA in the laboratory diagnosis of HSK, taking into account both epithelial and stromal keratitis cases.

Interaction of herpes simplex virus with mononuclear phagocytes is dependent on the differentiation stage of the cells.

The interaction of herpes simplex virus (HSV) with mononuclear phagocytes (MP), i.e. monocytes and macrophages, is of importance for the pathogenesis of HSV infections. MP are known to play a significant role in the cellular defence against infections with HSV, but it has also been shown that HSV-1 affects MP. The infection of these cells at different stages of differentiation has different outcomes, and may result in the alteration of important cellular functions. HSV-1 inhibits the morphological differentiation of human monocytes, and this inhibition occurs in spite of the fact that human monocytes are non-permissive to HSV-1. We have studied the effect of HSV infection of monocytes and macrophages on production of essential cytokines and related this effect to the reproduction of the virus. Blood-derived MP were cultured in vitro and inoculated with HSV at different stages of differentiation. Replication of the virus was measured by infectivity titration, detection of HSV antigens by immunofluorescence and detection of HSV-specific mRNA. In monocytes, no viral replication and no production of late protein was seen. HSV IE gene was transcribed in monocytes from some donors, but not from others. In macrophages, virus replicated, but less efficiently than in fully permissive fibroblast cells. The production of IL-1 beta, IL-6 and TNF-alpha in both non-permissive monocytes and permissive macrophages was assayed both at the transcriptional level, as mRNA, and as protein released from the cells. Production of cytokines by MP was affected by HSV-1. The level of cytokine mRNA and cytokine protein did not correspond for all cytokines, which may suggest that translational regulation and/or cytokine inhibitors are important in the regulation of the cytokine response. The cytokine modulation, both at the transcriptional level and measured as biological activity, was different in monocytes and macrophages, and varied between different donors. Our results indicate a relation between permissiveness and cytokine response in mononuclear phagocytes infected with HSV-1. Such a relation may be of importance to both intrinsic and extrinsic defence mechanisms of MP against HSV-1. Our study also demonstrates that even the functions of non-permissive cells such as blood-derived monocytes may be affected by viral infections.

Spin-amplified culture followed by enzyme immunoassay for detection of herpes simplex virus in patient specimens: a comparative study

In a comparative study it was found that a combination technique of spin-amplified culture and detection of herpes simplex virus (HSV) antigen in 48-h incubated cell culture lysate by a HSV antigen detection enzyme immunoassay kit (Dupont Herpchek) detected the largest number (227) of confirmed HSV positives when compared to standard cell culture (191) and direct Herpchek (146) on the same 415 clinical specimens.

The reliability of serological tests for the diagnosis of genital herpes: a critique

The reliability and limitations of the currently used routine tests for herpes simplex virus type 2 (HSV-2) antibody in Australia are reviewed. Six case reports illustrate the clinical dangers of overinterpretation of the currently available kits and the need for a readily available specific HSV-2 antibody test. In Sydney, HSV-2 causes approximately 85% of primary genital herpes and > 95% of recurrent genital herpes. Due to the extensive serological cross-reactivity between HSV-1 and HSV-2, currently available "type specific" commercial assays cannot reliably distinguish between the 2. Isolation of herpes simplex virus (HSV) or detection of HSV antigen in vesicle fluid is the preferred diagnostic test but may be overlooked or patients may have no visible lesions. The only accurate techniques for detecting HSV-2 specific antibody are the Western blot assay and an enzymatic immunoassay using glycoprotein G (gG-2), a component of the HSV-2 envelope. These tests, which still are restricted to research laboratories can be used to accurately identify people with previous exposure to HSV-2 (IgG) or to diagnose primary infection where virus isolation has not been performed or is impossible. Current commercially available antibody tests may have extensive cross-reactivity.

Rapid in vivo reactivation of herpes simplex virus in latently infected murine ganglionic neurons after transient hyperthermia.

A rapid and physiologically relevant hyperthermia-based induction procedure has been utilized to develop an in vivo model of induced herpes simplex virus (HSV) reactivation in outbred Swiss Webster mice. This procedure was found to efficiently reactivate latent virus from both trigeminal and lumbosacral ganglia. Examination of the time between hyperthermia and virus production demonstrated that detectable levels of infectious virus were present in ganglia as soon as 14 h posttreatment, with peak percent recoveries at 24 h. These data indicated that the switch from latent to active viral gene transcription occurred rapidly following treatment. Immunohistochemical staining for HSV type 1 antigens revealed rare antigen-positive ganglionic neurons 24 h postinduction. HSV antigens were not detected in any other cell type, and lateral spread of the infection was not observed. This is the first report of the detection of HSV antigens in vivo following induced reactivation in the intact nervous system and demonstrates that the neuron is the site of infectious virus production. In addition, our data strongly suggest that at least some neurons in which HSV antigens are detected during reactivation do not survive. Because the temporal and spatial characteristics of HSV reactivation have been clearly defined, this model is uniquely suited for the molecular dissection of the reactivation process.

Replacement of special enzyme immunoassay transport medium by a standard viral transport medium in the herpcheck herpes simplex virus antigen detection test

A new direct herpes simplex virus antigen enzyme immunoassay (EIA) uses a special EIA transport medium (ETM) for transport of herpes simplex virus (HSV) specimens. As ETM lyses the virus precluding culture and typing, we evaluated the relative performance of this EIA when performed on specimens transported in either ETM or a standard viral transport medium (VTM). These EIA results were also compared to cell culture performed on specimens transported in VTM (VTM-CC) and direct rapid inoculation into cell culture (CC). Based on all confirmed positives, by any test, the sensitivities for CC was 97% ( 66 68 ), for VTM-CC 91% ( 62 68 ), for ETM-EIA 97% ( 66 68 ), and for VTM-EIA 93% ( 63 68 ). It appears that VTM may be a slightly less desirable substitute than ETM in the performance of EIA. However, VTM-EIA is certainly as sensitive as cell culture performed on VTM.

Evaluation of a latex agglutination test for herpes simplex virus.

We evaluated a new commercial latex agglutination test for herpes simplex virus antigen detection (Virogen Herpes Slide Test, Wampole Laboratories, Cranbury, N.J.). In testing of eluates from 100 lesion swabs, mostly from genital sites, the following test parameters were ascertained: sensitivity, 73%; specificity, 89%; positive predictive value, 89%; negative predictive value, 72%. Testing was also performed on 160 supernatants from 153 cell cultures inoculated with clinical samples. In this application, sensitivity and specificity were each 100%, provided supernatants were sampled for testing when the cytopathic effect involved at least 25% of the cell monolayer.

Application of immunoperoxidase staining in the cytodiagnosis of herpes simplex keratitis

In corneal scraping smears from 13 patients with clinically suspected herpes simplex keratitis (HSK), HSK is demonstrated by means of peroxidase-antiperoxidase (PAP) technique with antisera to herpes simplex virus (HSV) in Papanicolaou-destained cellular samples. The staining for HSV antigen was present in seven cases of corneal scraping smears with superficial keratitis (dendritic and geographic ulcers) while six cases of stromal keratitis (deep keratitis) failed to show HSV antigen except in one case. Specific antigen for HSV was predominantly present in the cytoplasm rather than in the nucleus. Immunoreactions were negative with HSV antisera in patients with other infections and in those in a normal control group. Using the PAP technique, detection of HSV antigen in corneal scraping smears was of great value in the diagnosis of HSK, especially in cases of superficial keratitis.

Recurrent genital herpes simplex virus infection in guinea pigs.

After recovery from initial genital herpes simplex virus (HSV) infections, female guinea pigs developed spontaneous recurrent infections characterized by discrete erythematous or vesicular herpetic lesions on the external genital skin. HSV type 2 (HSV2) caused significantly more recurrent infections in guinea pigs than did HSV type 1 (HSV1). HSV2-infected animals demonstrated a significant decline in frequency of recurrences over time. The viral nature of the recurrent lesions was confirmed by recovery of infectious HSV, detection of HSV antigen, and histologic examination. Latent HSV2 could be demonstrated in dorsal root ganglia and external genital skin after recovery from the primary infection. Recurrent genital HSV infection in the guinea pig shares many features with recurrent genital herpes in humans and provides a model for studying the relationship between latency and recurrences and for exploring methods for control of recurrent disease.