间接免疫荧光法检测生殖器疱疹病毒

目的:评价间接免疫荧光试验(IFA)在生殖器疱疹(GH)诊断中的应用价值。方法:采用以单纯疱疹病毒(HSV)型共同性单克隆抗体为夹心的IFA法,检测了120例临床诊断为GH患者皮疹中的HSV,并与病毒培养法进行比较。结果:IFA检测HSV的总阳性率为85.8%,高于病毒培养法的阳性率(70.8%,χ2=12.04,P<0.01)。两种方法检测GH水疱内的HSV阳性率分别为93.3%和90.0%,无明显差异(χ2=1.96,P>0.05);而检测糜烂和结痂性皮疹内的HSV时,IFA的阳性率分别为92.6%和69.4%,均分别高于病毒培养法(75.9%,χ2=5.82,P<0.05;47.2%,χ2=14.17,P<0.01)。结论:IFA法具有简单、快速、敏感性高的优点,适于检测GH患者皮疹内HSV,有临床实用价值。     

An evaluation of near patient tests for detecting herpes simplex virus type-2 antibody

OBJECTIVES: To examine the "in use" test characteristics of the POCkit "near patient" HSV-2 rapid test for the detection of HSV-2 IgG antibodies for use in the clinic. This test relies on a visual interpretation of the result. METHODS: 2093 serum samples, 229 from UK and 919 from Italian genitourinary medicine clinic patients and 945 from obstetric and gynaecology clinic patients in Italy were tested. Tests were carried out according to manufacturers' protocol in the United Kingdom and Italy. Three readers independently recorded a score for each test carried out and the results were compared. RESULTS: In the UK study, the three readers disagreed on the result on 5.2% of tests. In the Italian study, there was disagreement in 10.2% of tests. CONCLUSIONS: This study has demonstrated a problem in the subjective nature of the interpretation of the POCkit HSV-2 test. It highlights the need for adequate training of clinic staff and the need for clinics to adopt policies of quality assurance and ongoing monitoring which will ensure the validity and accuracy of this clinic based test.     

Rapid diagnostic tests for cutaneous eruptions of herpes simplex.

Comparison was made of the results of virus isolation, indirect immunofluorescent staining (IF-test) and Tzanck smears from 32 patients with cutaneous eruptions clinically diagnosed as herpes simplex. Herpesvirus hominis was isolated from 21 patients; IF-test was positive in 22 patients; Tzanch smears were positive in 20 patients. 23 to 25 of the 25 patients in whom one of the diagnostic tests was positive could be identified by a combination of any two of the diagnostic methods employed. No false-positive reactions were observed for IF-test or Tzanck smear.     

Development and evaluation of a monoclonal antibody inhibition enzyme linked immunosorbent assay to diagnose syphilis.

A highly specific inhibition enzyme linked immunosorbent assay (ELISA) using murine monoclonal antibodies to treponemes has been developed to diagnose syphilis. The monoclonal antibodies used in this study were reactive to antigens of both Treponema pallidum and Treponema pertenue and not to antigens of non-pathogenic treponemes. Inhibition of the binding of monoclonal antibody to the treponemal antigens was successful with serum antibodies of patients with syphilis in an inhibition ELISA using monoclonal antibodies raised against T pallidum antigens with molecular weights of 42 and 47 kilodaltons. In contrast, the binding of monoclonal antibodies obtained by immunising mice with treponemal membrane protein TmpB, derived from recombinant DNA was not inhibited by serum antibodies from patients with syphilis. The sensitivity of the inhibition ELISA using monoclonal antibody against the 47 kilodalton T pallidum antigen was 93% in 58 serum samples from patients with untreated syphilis. The sensitivity was 79% if the monoclonal antibody against the 42 kilodalton T pallidum antigen was used. By a combination of the test results obtained in these two inhibition assays a sensitivity of 97% in the 58 serum samples from untreated patients and 64% in 64 from treated patients was obtained. The specificity of the inhibition ELISA performed with either monoclonal antibody was 100% in 500 serum samples from non-infected people. The specificity in 432 non-infected patients attending a sexually transmitted disease clinic was 98.8% for the monoclonal antibody against the 42 kilodalton antigen, 99.5% for the monoclonal antibody against the 47 kilodalton antigen, and 98.4% for the combined antibodies. The sensitivity and specificity of the inhibition ELISA using the combination of test results obtained by the application of the monoclonal antibodies against the 42 kilodalton treponemal membrane protein, TmpA, and against the 47 kilodalton T pallidum antigen were comparable with those of the Treponema pallidum haemagglutination assay (TPHA) and the fluorescent treponemal antibody absorption (FTA-ABS) test for diagnosing early untreated disease. The inhibition ELISA offers the potential for additional confirmation of early untreated syphilis. Its use for confirming late untreated syphilis is still under investigation. The test is highly specific for pathogenic treponemes and does not need sorbens.     

Ocular herpes simplex virus infections

Eye infection with herpes simplex virus is the single most common cause of corneal blindness in the United States and other industrialized countries. It occurs as often in developing countries. Herpetic eye disease presents a unique set of clinical problems, and there is considerable controversy even among knowledgeable ophthalmologists on the management of this disease.Like herpes simplex infections elsewhere, ocular herpetic infections are usually recurrent. While the disease often attacks the epithelial tissues of the lids, conjunctiva, and cornea, it can also involve the connective tissue of the cornea and interior structures of the eye. Most of the herpes isolates from the eye have been type 1, with only a low percentage of type 2 isolates.^1,2     

Enzyme linked immunosorbent assay for detecting antibody to Trichomonas vaginalis: use of whole cells and aqueous extract as antigen.

An enzyme linked immunosorbent assay (ELISA) for detecting antibody to antigenic Trichomonas vaginalis macromolecules has been identified using whole cells or an aqueous protein extract as antigen. The test was developed under optimum conditions using serum samples from experimental animals. The sensitivity of the ELISA was equal to or greater than that obtained by radioimmunoprecipitation and electrophoresis-fluorography techniques. The ELISA was capable of assessing antibody responses during the development of lesions in animals inoculated subcutaneously and it reproducibly measured the individual classes immunoglobulins directed at T vaginalis. The colorimetric assay was also suitable for showing cross reactivity between trichomonal species as well as between different strains of T vaginalis. Conditions established for monitoring antibody to trichomanads in immunised rabbits or infected mice were equally effective for human materials, such as serum or vaginal washes. Serum from experimental animals or infected people showed high concentrations of IgG, IgA, and IgM antibody to trichomonads. Only antibodies of the IgG and IgA class were detected in vaginal washes from women with acute trichomoniasis. No IgE antibody to trichomonads was found under a variety of conditions in serum samples from patients or experimental animals.     

Duplex real-time polymerase chain reaction assay for detection and quantification of herpes simplex virus type 1 and herpes simplex virus type 2 in genital and cutaneous lesions

BACKGROUND: A sensitive and specific method for detecting herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) is important for diagnosing genital and cutaneous infections. GOAL: The goal of this study was to compare quantitative real-time polymerase chain reaction (qPCR) with virus culture for diagnosis of genital and cutaneous HSV-1 and HSV-2. STUDY DESIGN: A duplex qPCR system for quantification of DNA from HSV-1 and HSV-2 was developed. Duplicate swabs for PCR and virus culture were collected from 89 patients attending our sexually transmitted infection and dermatology clinic. RESULTS: The duplex qPCR had a linear measure interval of 10-10 copies/mL. The detection limit was between 1 and 5 copies per reaction. qPCR detected HSV in 57 (64%) specimens and virus was isolated in 45 (50%) cases. First-episode infections showed higher viral quantities with a median value of 4.2 x 10 copies per reaction compared with recurrent infections with 1.0 x 10 (P = 0.0002). HSV-1 was more likely to be the cause of first-episode genital infections (72%), and HSV-2 of recurrent and atypical genital manifestations (73%). CONCLUSION: Real-time PCR is a sensitive method for diagnosing genital herpes, and the duplex format is convenient for typing. The method increased the detection rate by 27% compared with virus culture.     

An IgM capture enzyme linked immunosorbent assay to detect IgM antibodies to treponemes in patients with syphilis.

A new IgM capture enzyme linked immunosorbent assay (ELISA) was compared with the 19S(IgM) fluorescent treponemal antibody absorption (19S(IgM)FTA-ABS) test for detecting IgM antibodies to treponemes. Serum samples from 180 people, 109 with various stages of untreated syphilis, 45 with treated syphilis, and 26 non-infected, were investigated. In all diagnostic groups of syphilis the reactivity of the IgM capture ELISA was similar to that of the 19S(IgM)FTA-ABS test except in untreated neurosyphilis, for which the IgM capture ELISA was significantly less sensitive. The IgM capture ELISA was very sensitive in congenital (100%, 5/5) and primary (82%, 18/22) syphilis, but less sensitive in secondary (60%, 12/20), latent (53%, 16/30), neurosyphilis (34%, 11/32), and treated (11%, 5/45) syphilis. False positive IgM capture ELISA results were not found in five people who gave false positive Venereal Disease Research Laboratory (VDRL) reactions or in 21 neonates born to mothers adequately treated for syphilis before or during pregnancy. This indicated that the IgM capture ELISA was very specific. The course of antitreponemal IgM reactivity after treatment of early infectious syphilis was followed up in six patients. The quantity of IgM antibody declined in nearly all patients after treatment, but still remained detectable in five patients up to six months after treatment. In contrast, non-treponemal antibodies measured by the VDRL test disappeared in four out of six patients within five months from starting treatment. In conclusion, the IgM capture ELISA may be useful for easy and sensitive detection of IgM antibodies to treponemes in patients with congenital and primary syphilis. A positive test result in these cases indicates that patients should receive treatment if they have not been treated recently. The test is not, however, recommended to replace the VDRL test to monitor patients treated for syphilis.     

Rapid detection of glycoprotein G gene for the diagnosis and typing of herpes simplex virus infection in genital herpes

OBJECTIVE: To develop a new, rapid, and convenient technique for the diagnosis and typing of herpes simplex virus (HSV) in genital herpes (GH). METHODS: Using samples from skin vesicle fluid and urogenital mucosal swabs of subjects with GH, conventional polymerase chain reaction (PCR) (directed to polymerase gene: PCRpG) were compared with a newly developed PCR (directed to HSV glycoprotein gene: PCRgG). Both PCR methods were compared with virus isolation culture (VI) with indirect immunofluorescent staining (IIF). RESULTS: 80 samples from 40 GH patients (25 males) were tested. Positive results were seen in 52.5% (42/80) using PCRgG compared with 40% (32/80) by VI. Most of PCRgG positive samples (95.1%) were caused by HSV-2 infection. In samples from healing lesions, HSV was detected more often by PCRgG, than by VI. The results of typing by PCRgG and IIF were highly consistent. CONCLUSION: PCRgG is more sensitive than VI and PCRgG in detecting HSV in urogenital samples from subjects with GH. PCRgG is a convenient technique for the rapid detection and typing of GH.     

Detection of herpes simplex virus antigen with an enzyme immunoassay and direct immunofluorescence

A total of 464 clinical specimens from patients with extragenital and genital lesions and from the cervix of asymptomatic women were examined for the presence of Herpes-simplex-virus antigen by using a commercially available enzyme immunoassay Kit (ELISA, Dakopatts) and a direct immunofluorescence technique with monoclonal antibodies (Syva-Merck). The detection rate was compared to the isolation rate of the tissue-culture technique. In extragenital lesions the sensitivity of the ELISA technique and immunofluorescence was determined to be 78.9% and 80% and in genital lesions 46.2%. In cervical clinical specimens, the sensitivity of both techniques was determined to be 5.7% and 15.4%. An HSV infection, negative in tissue culture, was diagnosed in 22 and 27 cases, respectively, using ELISA and immunofluorescence. This diagnosis was confirmed in 21 and 22 cases by the other immunological technique. For this reason, it can be assumed that in some cases direct immunological techniques can be more sensitive than the tissue culture technique. With regard to asymptomatic and manifest infections, reliable results are only obtainable in cases of manifest disease with blisters or scabs.     

Rapid detection of cutaneous herpes simplex virus infection with the polymerase chain reaction

A simple and specific method for detecting herpes simplex virus infection in routinely processed paraffin-embedded biopsy specimens is described. DNA is extracted from paraffin blocks, and subjected to DNA amplification with the polymerase chain reaction. After 40 rounds, an amplified band can be detected after agarose gel electrophoresis and ethidium bromide staining. This band is specific for herpes simplex virus, because tissues infected with related viruses do not give this amplified band. We have been able to detect viral DNA in small punch skin biopsies with this procedure, which can take as little as 6 h.     

Proctitis and herpes simplex virus in homosexual men.

In a study of the prevalence of rectal infection with herpes simplex virus (HSV) in a group of homosexual men the magnified appearance of the rectal mucosa correlated with isolation of the virus. HSV was isolated from rectal material from five of 77 men with proctitis of unknown cause but from none of 44 control patients without proctitis; two of four men with HSV proctitis were asymptomatic. Thus, the magnified rectal mucosal image, showing severe congestion, haemorrhage, and pus, appears to be a sensitive indicator of the presence of HSV proctitis.     

Management of acyclovir-resistant herpes simplex virus

In immunocompetent patients, HSV is controlled rapidly by the human host's immune system, and recurrent lesions are small and short lived. When treated with antiviral agents, these patients rarely develop resistance to these drugs. In contrast immunocompromised patients might not be able to control HSV infection. Thus, frequent and severe reactivations are often seen and might lead to fatal herpetic encephalitis or disseminated HSV infection. Treatment in these patients is limited because immunocompromised hosts often develop severe herpes disease refractory to antiviral drug therapy. It is therefore imperative that physicians develop regimens to deal with both receptive and refractory HSV disease. The following treatment protocol (modified from Balfour and colleagues) might serve as a guide until further investigation of new drugs is performed. In all patients standard oral ACV therapy should be initiated at a dose of 200 mg orally, five times a day for the first 3 to 5 days. Prior to treatment, cultures the lesions should be obtained to verify HSV etiology. If the response is poor, the dose of oral ACV should be increased to 800 mg five times a day. If no response seen after 5 to 7 days, it is unlikely that the lesion will respond to intravenous ACV (or chemically and structurally related drugs such as VCV or famciclovir), so an alternative regimen must be assigned. First, repeat cultures for vital, fungal, and bacterial pathogens must be performed. In addition, ACV susceptibility studies should be ordered, if available. If the mucocutaneous lesion is accessible for topical treatment, TFT (as ophthalmic solution) should be applied to the area three to four times a day until the lesion is completely healed. If the lesion is inaccessible or if the response to TFT is poor, therapy with intravenous foscarnet should be given for 10 days or until complete resolution of the lesions. The dosage of foscarnet should be 40 milligrams per kilogram three times per day or 60 milligrams per kilogram twice daily. If foscarnet fails to achieve clinical clearing, consideration should be given to use of intravenous cidofovir (or application of compounded 1% to 3% topical cidofovir ointment). Vidarabine is reserved for situations in which all of these therapies fail. If lesions reoccur in the same location following clearing, the patient should started on high-dose oral ACV (800 mg, five times daily) or intravenous foscarnet (40 mg/kg tid or 60 mg/kg bid) as soon as possible. When lesions occur in a different location, the patient should be treated initially with standard doses of oral ACV (200 mg, five times daily) and the above protocol should be followed should there be clinical failure. In the future, new treatment options for patients with documented HSV resistance will be important in reducing the clinical impact of HSV.     

Medical application of herpes simplex virus

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) are important human pathogens that cause a variety of diseases from mild skin diseases such as herpes labialis and herpes genitalis to life-threatening diseases such as herpes encephalitis and neonatal herpes. A number of studies have elucidated the roles of this virus in viral replication and pathogenicity, the regulation of gene expression, interaction with the host cell and immune evasion from the host system. This research has allowed the development of potential therapeutic agents and vectors for human diseases. This review focuses on the basic functions and roles of HSV gene products and reviews the current knowledge of medical applications of genetically engineered HSV mutants using different strategies. These major HSV-derived vectors include: (i) amplicons for gene delivery vectors; (ii) replication-defective HSV recombinants for vaccine vectors; (iii) replication-attenuated HSV recombinants for oncolytic virotherapy.