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.     

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.     

Transport Media for Herpes Simplex Virus Types 1 and 2

An evaluation was made of the recovery rate of herpes simplex virus (HSV) type 1 or 2 from 197 clinical specimens obtained in two or three charcoal transport media: Leibovitz viral transport medium, a modified Leibovitz-Emory medium (LEM), in which agarose was used instead of agar, and Amies bacterial transport medium. The specimens were stored and shipped for 1 to 19 days in these media at ambient temperature or in Hanks buffered-salt solution in dry ice. The results indicate that the LEM was most effective, particularly in the recovery of HSV type 2 from clinical specimens held at ambient temperature. In vitro and in vivo studies in genitally infected mice corroborated the observations obtained with human clinical specimens. The availability of transport media which can be used for shipment at ambient temperature offers clinicians easier accessibility to laboratory confirmation and antigenic typing of HSV from suspect herpetic infections.     

Hemagglutination-Inhibition Method and Immunofluorescence Staining with Venezuelan Equine Encephalomyelitis Virus

Hemagglutination and fluorescent antibody (FA) are compared for the direct detection of virus devoid of host cells. A determination was made of the minimal number of tissue plaque-forming units of Venezuelan equine encephalomyelitis virus that could be detected by the hemagglutination technique. Similar concentrations of the virus in bovine albumin borate saline, Brain Heart Infusion broth (Difco), and demineralized water were tested by the FA technique. Somewhat higher concentrations of the virus in bovine albumin borate saline were used in the hemagglutination-inhibition test. The quantitative hemagglutination procedure employed for these studies was carried out at 37 C for 75 min with variations in concentration of goose red cells. As a result of lowering the red cell concentration, smaller concentrations of virus were detected. The direct FA staining procedure applied to slide preparations containing known numbers of tissue culture plaque-forming units of virus was negative. Adsorbed viral antigen on agglutinated goose erythrocytes was visualized by direct and indirect FA techniques.     

Diagnosis of Genital Herpes by Polymerase Chain Reaction Amplification

A new polymerase chain reaction (PCR) method employing type-specific primers and probes was applied to 114 clinical specimens obtained from 58 female patients with genital lesions or who had a history of genital herpes. Ten and 15 specimens, respectively, were positive for herpes simplex virus (HSV)-1 and HSV-2 by cell culture. All of 10 culture-confirmed HSV-1 cases and 11 of 15 (73%) culture-confirmed HSV-2 cases were identified by PCR. Although there were several cases with discrepancy between cell culture and PCR for HSV-2, the results suggest that this PCR procedure could be applied to clinical specimens from the female genital tract.     

Rapid Detection of Salmonella Microcolonies by Fluorescent Antibody

A microcolony fluorescent-antibody (FA) procedure for detecting salmonellae was compared to the usual direct FA procedure on 304 environmental, food, and feed samples. The microcolony FA test detected all of the specimens found positive by culture, whereas the direct FA missed 3.1% of them. Both FA tests revealed stained organisms in some of the culturally negative specimens. The microcolony FA test has several advantages over the direct FA test: ease of examining the smears, elimination of the fluorescent background material, and increased sensitivity.     

Diagnostics for herpes simplex virus: is PCR the new gold standard?

Herpes simplex virus (HSV) is one of the most common, yet frequently overlooked, sexually transmitted infections. Since the type of HSV infection affects prognosis and subsequent counseling, type-specific testing to distinguish HSV-1 from HSV-2 is recommended. Although PCR has been the diagnostic standard for HSV infections of the central nervous system, until now viral culture has been the test of choice for HSV genital infection. However, HSV PCR, with its consistently and substantially higher rate of HSV detection, will likely replace viral culture as the gold standard for the diagnosis of genital herpes in people with active mucocutaneous lesions, regardless of anatomic location or viral type. Alternatively, type-specific serologic tests based on glycoprotein G should be the test of choice to establish the diagnosis of HSV infection when no active lesion is present. Given the difficulty in making the clinical diagnosis of HSV, the growing worldwide prevalence of genital herpes and the availability of effective antiviral therapy, there is an increased demand for rapid, accurate laboratory diagnosis of patients with HSV.     

Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids

A fully automated multiplex real-time PCR assay—including a sample process control and a plasmid based positive control—for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88–258) copies/ml for HSV1, 171 (CI 95%, 148–194) copies/ml for HSV2 and 84 (CI 95%, 5–163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements.     

炭凝集试验快速检测呼吸道合胞病毒的研究

用炭凝集试验（ＣＡＴ）检测呼吸道合胞病毒（ＲＳＶ）,结果表明该法是一种简便、快速、特异的诊断方法。用ＣＡＴ对１６株ＲＳＶ和８株其它病毒做试验,结果仅ＲＳＶ凝集,而其它病毒均阴性。用该法与细胞培养法检测８３份临床呼吸道感染幼儿鼻咽吸出物,结果ＣＡＴ法阳性率为６９．８８％（５３／８３）,细胞培养法为３９．７５％（３３／８３）,两者阳性检出率相差极显著。阻断试验证明ＣＡＴ是高度特异的。结果证明ＣＡＴ具有较高的敏感性与特异性,可用于临床ＲＳＶ标本的快速检测。     

The development and testing of test systems for the determination of herpes simplex virus and cytomegalovirus antigens by lanthanide immunofluorescence analysis

On the basis of the lanthanide immunofluorescent assay (LIFA) test systems for the determination of herpes simplex virus (HSV-LIFA) and cytomegalovirus (CMV-LIFA) antigens have been developed. The test system HSV-LIFA includes the sandwich of monoclonal antibodies (McAb) HSV A.3.3. or B-11 to type 2 HSV, strain BH; the test system CMV-LIFA includes the sandwich of rabbit McAb to CMV, strain AD 169. The approbation of the test systems has revealed that they insure the specific detection of HSV and CMV antigens in clinical specimens (urine, blood, liquor, saliva), the LIFA results well correlating with the data on the isolation of viruses in cell cultures, with the results obtained by other diagnostic methods and with clinical manifestations of diseases. LIFA has been shown to be more sensitive than the enzyme immunoassay.     

A rapid and automated colorimetric assay for evaluating the sensitivity of herpes simplex strains to antiviral drugs

A rapid and sensitive colorimetric assay has been developed to evaluate the sensitivity of herpes simplex viruses (HSV) to antiviral agents. The chessboard titration of viruses and antiviral drugs and the automatic reading and analysis of the data allows objective and accurate results to be rapidly obtained. Virus sensitivity was expressed as an ED50 value which was the concentration of drug (micrograms/ml) reducing viral cpe by 50%. The ED50 values of antiviral drugs [acetylguanosine (ACV), idoxuridine (IDU), deoxycytidine (IDC) and bromovinyl deoxyuridine] for several HSV reference strains were determined and the method was then applied to clinical specimens. The selection of ACV and IDU resistant mutants was performed on a cloned sensitive HSV 1 ocular strain. We observed cross-resistance between ACV and IDU for the mutants isolated. The resistance to thymidine-kinase-dependent antiviral agents varied in inverse ratio to the thymidine kinase activity induced by HSV strains.     

The detection of the herpes simplex virus 1 and 2 antigens in clinical specimens by using monoclonal antibodies

The possibility of using monoclonal antibodies (McAb), obtained earlier, for the detection of herpes simplex virus (HSV) in clinical specimens taken from sick and infected persons was studied. The examination of 90 persons revealed that the mixture of McAb 4A and 2C could effectively detect the presence of HSV antigen in the indirect immunofluorescence assay (IFA) directly in cells contained in cytological preparations (smears, scrapes, impressions) obtained from different organs of patients. The search of optimum combinations of McAb for the detection of HSV antigens by the method of the solid-phase enzyme immunoassay (EIA) was carried out. This study, made on purified HSV used as an experimental model, revealed that the maximum sensitivity could be achieved with the use of two McAb (4f6 and 7c4) out of three McAb (4f6, 7c4 and 3d10). The approbation of both variants of EIA on clinical specimens taken from 99 patients (blood clots, seminal fluid, scrapes of cervical canal cells, peripheral blood lymphocytes) showed that the addition of McAb 3d10 made it possible to detect 8 more positive specimens. 754 specimens from 337 patients were studied with the use of McAb-based EIA, and in 204 of these patients (61%) HSV antigen was detected. The results obtained with the use of our McAb were compared with the data obtained with certified commercial test systems. The coincidence of the EIA data with those obtained with the use of the Murex Wellcozyme HSV test system (UK) was registered in 75% of cases (in 15 out of 20 cases). The coincidence of the IFA data with those obtained with the use of the Sanofi test system (France) was observed in all 19 cases (100%).     

In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture.

The F strain of herpes simplex virus type 1 (HSV-1) was tested for its ability to produce lytic or nonproductive infection in squamous epithelial cells cultured in a three-dimensional organotypic tissue culture. For the tissue culture, we used HaCat cells (immortalized skin keratinocytes) and normal fibroblasts derived from the skin. The cultures were infected with HSV-1 (5 PFU) either when the epithelial cells had grown as a monolayer with a confluence of 80% on the collagen fibroblast gel or 30 min after lifting of the epithelial cells into the air-liquid interface. The cultures were collected 1 week after inoculation. Typical cytopathic effects of HSV infection (ballooning and reticular degeneration with multinucleate giant cells) were seen only in those cultures in which the epithelial cells were infected before lifting. The presence of HSV was confirmed by DNA and RNA in situ hybridization and PCR. No morphological changes were found in cultures infected after lifting into the air-liquid interface. No infectious virus was recovered either from cells or culture supernatant. However, these cultures were positive for HSV DNA on PCR and showed expression of the LAT gene by in situ hybridization and Northern blot (RNA) hybridization. The present results indicate that both nonproductive and lytic HSV infection can be produced in vitro and the outcome of the infection depends on the time of viral inoculation in relation to epithelial maturation.     

Detection of herpes simplex and varicella zoster viruses in clinical specimens using direct immunofluorescence and cell culture assays

Patients (33 in toto) with a clinical diagnosis of herpes infections (simplex, zoster or chickenpox) were investigated for the presence of herpes simplex virus (HSV) and varicella zoster virus (VZV) in skin samples, using direct immunofluorescence and cell culture assays. Five patients with nonherpetic vesiculobullous disorders were included as negative controls. Of the 33 patients, nineteen (57.6%) were positive for HSV or VZV and fourteen (42.4%) were negative. Five controls were all negative for HSV or VZV. Of the nineteen positive patients, HSV was isolated from eight (42.1%) patients, by both direct immunofluorescence and cell culture assays. VZV was isolated from eleven (57.9%) patients, eleven (100%) by direct immunofluorescence assay, and six (54.5%) by cell culture assays. HSV was isolated from one patient clinically diagnosed as chickenpox (VZV), but otherwise the positive laboratory results were concordant with the clinical diagnosis. For epidemiological studies, atypical cases and immunocompromised patients the clinical diagnosis should be confirmed in the laboratory.     

An analysis of factors influencing the isolation rate of herpes simplex virus.

Attempts were made to improve the rate of isolation of herpes simplex virus (HSV) from clinical specimens by minimizing loss of virus infectivity during transportation and employing the most sensitive cells for isolation. Basical analyses using standard strains of type 1 and type 2 HSV indicated that virus titer decrease was marked even at low temperatures in environments free of proteinous stabilizer such as normal serum or tissue extract, negating the generally held concept that HSV is stable in distilled water. YLE (Earle-lactalbumin HYDROLYSATE-YEAST EXTRACT) medium containing 20% inactivated calf serum was determined to be a transport medium of choice, because degradation of suspended virus during storage and freeze-thawing was negligible and loss of virus during Millipore filtration was minimal. Special coating of the membrane could also be obviated by the use of this solution. In a cell susceptibility test using clinical specimens, secondary rabbit kidney (SRK) cells were the most sensitive, showing a quick development of cytopathic effect. Vero and RK-13 cells were the second best, whereas monkey kidney, HeLa and L cells were far less sensitive. A total of 136 specimens from suspected cases, sent by dermatologists, were tested using SRK cells, and 99 strains of type 1 and 15 strains of type 2 HSV were isolated. Excluding one case from which vaccinia virus was isolated, the isolation rate of HSV was 84.4%.     

A micro-neutralization system for detection of s-CRN antibody to herpes simplex virus

It was learned that the ordinary micro-neutralization system with herpes simplex virus (HSV) gave a composite result of the initial neutralization and the effect of antibody on subsequent growth of unneutralized virus. In the case of slow-reacting complement-requiring neutralizing (s-CRN) antibody, which was detected by incubating virus-serum mixtures at 4 C for 3 days before addition of complement, the titer obtained was lower than expected from the result of the plaque reduction test. This was thought ascribable to its low ability to prevent viral breakthrough caused by growth of unneutralized virus. This was overcome by adding an appropriate amount of hyperimmune antibody at 3 hr after addition of cells. The endpoint of s-CRN antibody so determined was but slightly lower than that obtained by the plaque reduction test. Early (1-week) rabbit sera, which were negative in the ordinary micro-neutralization test, titered 1:2,560 to 1:5,120 when tested by this method. When the 3-day sensitization in the cold was substituted by 5-hr incubation at 37 C, the titer obtained was 2 to 4-fold lower; in this case, however, the whole process could be finished within 3 days. Also, s-CRN antibody reactive with type 2 HSV in homologous and heterologous sera could be detected by the same method using type 1 hyperimmune serum as the additional antibody.     

Detection of herpes simplex and varicella-zoster virus DNA by field-inversion gel electrophoresis from clinical materials.

A simple method using field-inversion gel electrophoresis (FIGE) was applied to detect herpes simplex virus (HSV) and varicella-zoster virus (VZV) genomes in clinical specimens. The whole genomes of these viruses could be detected in small vesicle tissues by the FIGE method regardless of their clinical stages of skin lesions. And the sensitivity of the FIGE method was equivalent to that of an immunofluorescent (IF) method. These data indicated usefulness of the FIGE method to detect the whole genomes of HSV and VZV in clinical specimens.     

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.     

Typing foot-and-mouth disease virus by fluorescent antibody technique.

Typing of foot-and-mouth disease (FMD) virus was performed by the direct fluorescent antibody (FA) technique. Type-specific FA was prepared from the following two sorts of procedures: (1) FA against live virus (FA-live) was prepared from hyperimmune serum taken from guinea pigs having received live FMD virus. Then it was adsorbed with concentrated heterotype antigen. (2) FA against inactivated virus (FA-Inact) was prepared from antiserum taken from guinea pigs immunized with purified FMD virus inactivated with acetylethyleneimine. Seventeen strains of FMD virus (seven strains of type A, seven strains of type O, and three strains of thpe C) were used. Type-specific FMD virus antigen was detected distinctly from the monolayer of BHK cells infected with each type of virus and fixed in acetone, in spite of negative results obtained from the cells fixed in methyl alcohol. All the 17 strains were typed successfully by the implementation of these two FA methods.