The use of peptides from glycoproteins G-2 and D-1 for detecting herpes simplex virus type 2 and type-common antibodies.

BACKGROUND: identification and discrimination of latent herpes simplex virus (HSV) infection relies on antibody identification. The inclusion of synthetic peptides with HSV glycoproteins provides means for stable and discriminatory assays for population studies. OBJECTIVE: to determine whether virus-specific synthetic peptides might identify HSV type 2 (HSV-2) antibodies in the presence of the cross-reactive and more common HSV type 1 (HSV-1) antibodies. STUDY DESIGN: the capacity of synthetic peptides as HSV antigens was analyzed in enzyme immunoassay (EIA) using well characterized human serum cohorts. The HSV peptide assays were evaluated in comparison with two commercial HSV-2 assays. RESULTS: a combination of two C-terminal HSV-1 glycoprotein D (gD-1) peptides detected type-common HSV immunoglobulin G (IgG) with high sensitivity (95%) and specificity (93%). Peptides derived from the C-terminus of HSV-2 glycoprotein G (gG-2) had a high HSV-2 type-specificity. Inclusion of both gD-1 and gG-2 peptides gave a sensitivity for human anti-HSV-2 IgG that was similar to that of assays including different amounts of native gG-2. With western blotting as a standard, the sensitivity of the peptide assay ranged between 86% for HSV-2 seropositive persons and 61% for HSV-2 seroconverters. Addition of a small amount of native gG-2 to the peptide assay tended to increase the specificity. CONCLUSION: HSV gG and gD peptides show promise as type-specific and type-common HSV antigens. These peptides are more stable and reproducibly prepared than native or recombinant glycoproteins and may be considered for inclusion in future HSV serodiagnostic assays.     

Evaluation of new reagents for typing IgG to HSV-1 and HSV-2

Until recently, the lack of suitable type specific assays has hampered the serological diagnosis of herpes simplex virus (HSV) infections, due to the high crossreactivity between types 1 and 2. The aim of the present paper is the evaluation of new commercial methods for the detection of HSV-1 and HSV-2-specific IgG using glycoprotein G as antigen (BioElisa HSV-1 and BioElisa HSV-2), in their application to viral diagnosis and seroepidemiological studies. Eighty two serum samples from HSV recent infections (30 samples from 13 cases), normal children (28 samples), and patients attended in clinics for sexually transmitted diseases (STD) (24 samples from 20 patients) were studied by such methods and the results compared with those obtained by a conventional indirect ELISA, and by the complement fixation test. The methods gave a type specific identification of the antibody response in nine of the 13 HSV patients. Positive results for anti-HSV-2 IgG were obtained in four cases among the STD patients but in none among the normal children. Nineteen of the former and seven of the latter were positive for anti-HSV-1. Only one sample was reactive in the HSV-1 assay, and negative by the indirect ELISA. Type specific HSV-1 and HSV-2 assays may help the serological diagnosis of HSV infections, since they allow the correct characterization of the antibody response. Bearing in mind the high specificity of the method for HSV-2 IgG, it might be useful in screening of populations for anti-HSV-2 and especially in prevention of the neonatal HSV-2 infection.     

Evaluation of confirmatory strategies for detection of type-specific antibodies against herpes simplex virus type 2

In this study, the optimal combination of three commercial glycoprotein G-2 (gG-2)-based herpes simplex virus type 2 (HSV-2) type-specific enzyme-linked immunosorbent assays (Euroimmun anti-HSV-2 immunoglobulin G [IgG] ELISA [Eu2], Gull HSV-2-specific IgG ELISA [Gu2], and Radim HSV-2 IgG ELISA [Ra2]) and one gG-2-based HSV-2-specific immunoblot (Euroimmun anti-HSV-1/HSV-2 gG Western blot [EuW]) was determined with regard to diagnostic performance and cost efficiency. Two hundred fifty serum samples were included in this study, 194 of which were from female prostitutes. When a formal primary "gold standard" was defined based on majority agreement of the commercial tests, with EuW being decisive in stand-off situations, the sensitivity and specificity of the assays in the samples from prostitutes were as follows: Eu2, 100 and 89.22%; Gu2, 94.44 and 96.08%; Ra2, 61.18 and 95.10%; and EuW, 98.90 and 100%. The most cost-effective confirmatory strategy in the samples from prostitutes was screening with Eu2, retesting positive and equivocal samples with Gu2, and resolving the remaining discordant results with EuW (estimated additional costs per sample, 79.02%; sensitivity, 100%; positive predictive value, 96.81%). Applying a self-developed gG-2-independent assay to the discordant and concordant negative samples in the samples from prostitutes suggested that the primary gold standard may have missed six HSV-2-positive samples. In conclusion, confirmatory strategies based on commercial gG-2-dependent seroassays result in an increase in the specificity of HSV-2-specific serology. However, further improvement of the sensitivity of current HSV-2-specific serology may require the additional exploitation of the gG-2-independent type-specific antibody response.     

Type specificity of complement-fixing antibody against herpes simplex virus type 2 AG-4 early antigen in patients with asymptomatic infection.

We evaluated the type specificity of complement-fixing (CF) antibody against the AG-4 early antigen of herpes simplex virus (HSV) type 2 (HSV-2) by comparing a commercial AG-4 CF kit (Simplex-2; Gene Link Australia, Inc., Princeton, N.J.) with quantal microneutralization (MN) and absorption-Western blotting in testing sera from patients with and without a history of genital herpes. Sera characterized as HSV type 1 (HSV-1) or HSV-2 positive or negative by MN were selected and tested by CF, and those with discordant results were further analyzed for specific antibodies by absorption with HSV-1 or HSV-2 antigen and Western blotting with heterologous HSV proteins. A total of 34 of 42 (81%) sera HSV-2 positive by MN, 19 of 43 (44%) sera HSV-1 positive by MN, and 0 of 19 sera negative by MN were positive by CF. Absorption-Western blotting showed that 12 of 18 (67%) sera HSV-1 positive by MN but positive by CF had no HSV-2-specific antibody and that all 7 sera HSV-2 positive by MN but negative by CF had HSV-2-specific antibody. When MN and absorption-Western blotting data were combined to analyze patients with no history of genital herpes, 7 of 19 (37%) with no HSV-2-specific antibody were positive by CF, and 7 of 27 (26%) with HSV-2-specific antibody were negative by CF. The positive and negative predictive values for the CF test were 78 and 75%, respectively, in this group. The presence of antibody to the HSV AG-4 antigen does not discriminate sufficiently between HSV-1- and HSV-2-infected patients to be of value in predicting HSV-2 infection in the absence of symptomatic disease.     

Evaluation of five monoclonal antibody-based kits or reagents for the identification and culture confirmation of herpes simplex virus.

Five immunofluorescence (IF) kits or reagents (Bartels [Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.], Imagen [CellTech Diagnostics, Ltd., distributed by Analytab Products, Plainview, N.Y.], Ortho [Ortho Diagnostics Systems, Inc., Raritan, N.J.], Syva [Syva Co., Palo Alto, Calif.], Whittaker [Whittaker Bioproducts, Walkersville, Md.]) were evaluated for typing and laboratory confirmation of herpes simplex virus (HSV). Of 101 clinical isolates tested by each kit or reagent, results for 97 of them were in agreement. Identification of the four isolates with discordant results was performed by restriction endonuclease analysis of the viral DNA. The sensitivity and specificity of the Imagen and Bartels kits were 100%. For the Ortho, Syva, and Whittaker kits or reagents, the HSV type 1 (HSV-1) and HSV type 2 (HSV-2) sensitivities were 97.4 and 100%, 100 and 100%, and 97.4 and 100%, respectively, and the specificities were 100 and 97.4%, 100 and 92.4%, and 100 and 97.4%, respectively. There was one false-positive HSV-2 isolate identified by each of the Ortho and Whittaker kits or reagents. Three false-positive HSV-2 isolates occurred by staining with Syva, giving the erroneous indication of dual isolates. Several isolates stained with Imagen and Whittaker reagents displayed dull IF patterns. A dull green background occurred in ca. one-third of the HSV-2 isolates tested with the Ortho kit. The intensities of IF staining by the Bartels and Syva kits were satisfactory; however, the latter displayed a specificity of 92.7%. A total of 38 and 63 specimens were finally designated as HSV-1 and HSV-2, respectively. Identification of each isolate with the Bartels kit was consistently interpretable and is recommended as the typing and confirmatory assay of choice.     

地高辛标记探针在单纯疱疹病毒检测及分型中的应用

利用地高辛配基制备单纯疱疹病毒型特异性ＤＮＡ片段探针，对４４株单纯疱疹病毒（ＨＳＶ）临床分离株作了分型鉴定。结果显示２５株为ＨＳＶ－１，１９株为ＨＳＶ－２。比较研究发现，地高辛标记与同位素３２Ｐ中标记探针检测符合率为１００％，地高辛标记探针在灵敏性、特异性方面与同位素探针相同，对ＨＳＶ的分型率均达１００％。由于地高辛标记探针无放射性污染，能长期贮存及多次使用，适合在基层医院推广应用。     

Determination of herpes simplex virus type-specific antibodies by enzyme-linked immunosorbent assay.

We determined type-specific antibodies to herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) by an indirect enzyme-linked immunosorbent assay, using as antigens HSV-1 glycoprotein gC-1 and a HSV-2-specific polypeptide purified on affinity columns of monoclonal antibodies. All sera were initially screened for HSV antibodies by the enzyme-linked immunosorbent assay with a pool of Triton X-100-extracted antigens of HSV-1- and HSV-2-infected HEp-2 cells. The titer of HSV antibodies was predicted from a linear regression curve based on the absorbance of the initial 1:50 serum dilution. The sensitivity and specificity of the screening assay and of the assay for type-specific antibodies were established.     

Comparison of a monoclonal antibody-blocking enzyme-linked immunoassay and a strip immunoblot assay for identifying type-specific herpes simplex virus type 2 serological responses

Detection of herpes simplex virus type 2 (HSV-2)-specific antibodies by a monoclonal antibody (MAb)-blocking enzyme-linked immunoassay (EIA) was compared with detection by a strip immunoblot assay (SIA) in a sexually transmitted disease (STD) clinic population. The study population consisted of 1,683 genitourinary medicine clinic attendees (582 women and 1,101 men). Sera were tested for the presence of HSV-2 antibody by use of the blocking EIA, in which binding of the MAb AP-1 to HSV-2 glycoprotein G-2 (gG-2) is blocked by HSV-2-specific antibody. The Chiron RIBA HSV-1 and -2 strip immunoassay (SIA) utilizes HSV-1- and HSV-2-specific or cross-reactive antigens immobilized on nitrocellulose strips (HSV gB-1 and HSV gG-1 peptide bands specific for HSV-1 antibody, HSV-2 gG-2 band specific for HSV-2 antibody, and HSV gD-2 band cross-reactive for HSV-1 and HSV-2 antibodies). A total of 1,612 sera were tested by MAb-blocking EIA for HSV-2 antibody and by SIA for HSV-1 and HSV-2 antibodies. By EIA, 541 (33.6%) sera were positive for HSV-2 antibody and 1,068 sera were negative for HSV-2 antibody; 3 sera gave equivocal results. HSV-2 antibody was detected in 555 (34.4%) sera by SIA; 144 (26%) of these sera possessed only HSV-2 antibody, and 411 (74%) sera contained both HSV-1 and HSV-2 antibodies. SIA detected HSV-1 antibody in 1,155 (71.6%) sera; 744 (64%) of these sera contained HSV-1 antibody alone. Sixteen sera contained antibody against HSV but could not be typed by SIA. A total of 512 sera were positive for HSV-2 antibody by both the EIA and SIA. We concluded that the blocking EIA and SIA showed a high level of agreement in detecting HSV-2 antibody in this population. In contrast to the SIA, the blocking EIA is a useful tool for large epidemiological studies, though the SIA proved to be slightly more sensitive once sera with discrepant results were further tested.     

Comparison of a Monoclonal Antibody-Blocking Enzyme-Linked Immunoassay and a Strip Immunoblot Assay for Identifying Type-Specific Herpes Simplex Virus Type 2 Serological Responses

Detection of herpes simplex virus type 2 (HSV-2)-specific antibodies by a monoclonal antibody (MAb)-blocking enzyme-linked immunoassay (EIA) was compared with detection by a strip immunoblot assay (SIA) in a sexually transmitted disease (STD) clinic population. The study population consisted of 1,683 genitourinary medicine clinic attendees (582 women and 1,101 men). Sera were tested for the presence of HSV-2 antibody by use of the blocking EIA, in which binding of the MAb AP-1 to HSV-2 glycoprotein G-2 (gG-2) is blocked by HSV-2-specific antibody. The Chiron RIBA HSV-1 and -2 strip immunoassay (SIA) utilizes HSV-1- and HSV-2-specific or cross-reactive antigens immobilized on nitrocellulose strips (HSV gB-1 and HSV gG-1 peptide bands specific for HSV-1 antibody, HSV-2 gG-2 band specific for HSV-2 antibody, and HSV gD-2 band cross-reactive for HSV-1 and HSV-2 antibodies). A total of 1,612 sera were tested by MAb-blocking EIA for HSV-2 antibody and by SIA for HSV-1 and HSV-2 antibodies. By EIA, 541 (33.6%) sera were positive for HSV-2 antibody and 1,068 sera were negative for HSV-2 antibody; 3 sera gave equivocal results. HSV-2 antibody was detected in 555 (34.4%) sera by SIA; 144 (26%) of these sera possessed only HSV-2 antibody, and 411 (74%) sera contained both HSV-1 and HSV-2 antibodies. SIA detected HSV-1 antibody in 1,155 (71.6%) sera; 744 (64%) of these sera contained HSV-1 antibody alone. Sixteen sera contained antibody against HSV but could not be typed by SIA. A total of 512 sera were positive for HSV-2 antibody by both the EIA and SIA. We concluded that the blocking EIA and SIA showed a high level of agreement in detecting HSV-2 antibody in this population. In contrast to the SIA, the blocking EIA is a useful tool for large epidemiological studies, though the SIA proved to be slightly more sensitive once sera with discrepant results were further tested.     

Detection of herpes simplex virus in direct specimens by immunofluorescence assay using a monoclonal antibody.

A monoclonal antibody (MAb), designated CHA 437, was developed against herpes simplex virus (HSV). This MAb (isotype, immunoglobulin G2b K) reacted with HSV type 1 and HSV type 2. It showed no cross-reactivity with varicella-zoster virus, cytomegalovirus, or Epstein-Barr virus. Direct detection of HSV antigen in clinical specimens using indirect immunofluorescence with this MAb was compared with tissue culture isolation. For the 682 specimens tested, the direct specimen test gave a sensitivity of 84.6% and a specificity of 95.7%.     

Typing of herpes simplex virus with type-specific human immunoglobulin M in an indirect immunofluorescence assay.

Sera from nine individuals with suspected primary herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) infection were screened to identify those containing HSV type-specific immunoglobulin M (IgM). Selected sera were then utilized in an IgM-specific indirect immunofluorescent-antibody HSV-typing assay (patent pending). To evaluate the procedure, 29 HSV isolates were grown in cultures of continuous human amnion cells, fixed, and used as substrates for indirect immunofluorescence. Determination of virus type was based on intensity of fluorescence of the substrate with HSV-1- and HSV-2-specific antisera after staining with fluorescein-conjugated anti-human IgM. Typing of the isolates by restriction endonuclease digestion showed that of 29, 18 were HSV-2 and 11 were HSV-1. Results by IgM-specific indirect immunofluorescent-antibody assay were identical to those by restriction endonuclease digestion for 27 of the isolates; 2 isolates failed to replicate adequately in the test cells. The IgM-specific indirect immunofluorescent-antibody procedure appears to be a simple, rapid, and accurate technique which could be of use to clinical virology laboratories.     

Rapid detection of herpes simplex virus in clinical samples by flow cytometry after amplification in tissue culture.

Murine monoclonal antibodies (MAbs) against herpes simplex virus type 1 and 2 (HSV-1 and -2, respectively) nuclear antigens were used to identify cells infected with HSV-1 or -2 by indirect immunofluorescence in conjunction with flow cytometry after virus amplification of MRC-5 cell monolayers. The results indicate that MAbs Q1, Q2, and H-640 detect HSV-1- and HSV-2-infected cells, MAb SD-1 detects HSV-2- but not HSV-1-infected cells, and MAb 58-S detects HSV-1- but not HSV-2-infected cells. MAb Q1, which detects HSV-1- as well as HSV-2-infected cells, was used to detect HSV-infected cells after inoculation and overnight (16- to 20-h) incubation of MRC-5 monolayers with clinical samples suspected of containing HSV. In comparing the efficiency of flow cytometry with cytopathic effect (CPE) in tissue culture for detecting HSV in clinical samples, HSV was detected by flow cytometry in 77% of the cases where HSV was detected by CPE in tissue culture. In many cases, flow cytometry detected HSV from 1 to 3 days before HSV was detected by CPE.     

Isolation of the major herpes simplex virus type 1 (HSV-1)-specific glycoprotein by hydroxylapatite chromatography and its use in enzyme-linked immunosorbent assay for titration of human HSV-1-specific antibodies.

A 131,000 molecular weight herpes simplex virus type 1 (HSV-1) glycoprotein designated antigen number 6 (Ag-6) was previously shown to possess almost exclusively HSV-1-specific antigenic sites. Fused rocket and crossed immunoelectrophoresis of fractions obtained from hydroxylapatite chromatography of crude HSV-1 antigen (Triton X-100-solubilized, infected tissue culture cells) showed that a subfraction of Ag-6 could be separated from the other HSV antigens. Enzyme-linked immunosorbent assay with the isolated Ag-6 showed that sera from rabbits infected with HSV-1 and HSV-1 human antisera contained antibodies to Ag-6, whereas sera from HSV-2-infected rabbits and sera from patients with primary HSV-2 infections did not react with Ag-6. Enzyme-linked immunosorbent assay of 852 human sera for antibodies to HSV type-common glycoproteins, Ag-6, and HSV 2-specific antigens showed that 139 sera which reacted negatively with HSV type-common glycoproteins also did not react with Ag-6 with HSV-2 specific antigens. The 713 sera reacting positively to HSV type-common antigens either reacted with Ag-6 (328 sera) or with HSV-2-specific antigens (31 sera) or both (354 sera). This means that Ag-6 might be useful in large-scale human serology for the detection of past infection with HSV-1, irrespective of whether or not past infection with HSV-2 has occurred.     

ELISA for the detection of herpes simplex virus antigens in the cerebrospinal fluid of patients with encephalitis

An inhibition enzyme-linked immunosorbent assay (ELISA) for the detection of herpes simplex virus antigens in cerebrospinal fluid (CSF) has been developed. A Triton X-100 extract of herpes simplex virus type 1 (HSV-1) infected HEp-2 cells was used to coat wells of polyvinyl chloride plates. Rabbit anti-HSV-1 globulin served as the reference antibody and the CSF specimens were tested at a final dilution of 1:4. Positive results were obtained in CSF specimens from 11/18 (61%) neonates with HSV infection, 15/23 (65%) older individuals with HSV culture positive brain biopsies, and in 4/29 (14%) patients with culture negative brain biopsies. The assay was negative with CSF from 14 infants without HSV infections, from 30 patients with bacterial meningitis and 10 with cryptococcal meningitis. The test was positive in 10/21 patients within 10 days of onset, 11/14 within 11-20 days, and in 5/6 more than 20 days after onset of the herpetic infection. The overall sensitivity of the assay was 63% and the specificity was 95%.     

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay

While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques.     

Herpes simplex virus type-2 specific glycoprotein G-2 immunomagnetically captured from HEp-2 infected tissue culture extracts

Monoclonal antibody H1206 anti-HSV-2 gG-2 bound to tosylactivated paramagnetic Dynabeads (Dynal) has been used to isolate HSV-2 type-specific gG-2 from solubilized HEp-2 HSV-2 infected cell extracts. The immunomagnetically captured type-specific glycoprotein reacted strongly with monoclonal antibody H1206 and demonstrated a single band with apparent molecular weight of 100000 (100 kDa) and a doublet band with an apparent molecular weight of 60000-64000 (60-64 kDa). We observed the same exact banding pattern when monoclonal H1206 was immunoblotted with Helix pomatia lectin purified HSV-2 gG-2. The immunomagnetically purified gG-2 was unreactive to monoclonal antibody H1379 anti-HSV-1 gG-1 and four human HSV antibody negative sera. In addition, 20 human HSV antibody positive sera obtained from the Centers for Disease Control (CDC), Atlanta, GA, were used for the evaluation of our methodology. Immunoblotting of the human HSV antibody positive samples were in agreement with the CDC HSV serological designation. Sera characterized by reactivity to the immunomagnetically purified gG-2 in conjunction with Western blot has the potential to be used as a confirmatory serological test or to determine the accuracy of clinical serological immunoassays used to determine HSV-2 seropositivity.     

Type-Specific Identification of Anogenital Herpes Simplex Virus Infections by Use of a Commercially Available Nucleic Acid Amplification Test

Herpes infections are among the most common sexually transmitted infections (STI), but diagnostic methods for genital herpes have not kept pace with the movement toward molecular testing. Here, we describe an FDA-approved molecular assay that identifies and types herpes simplex virus (HSV) infections for use in routine clinical settings. Paired samples from anogenital lesions were tested using the BD ProbeTec HSV Q^x (HSVQ^x) system, HSV culture and, a laboratory-developed PCR assay. Family planning, obstetrics/gynecology (OB/GYN), or sexually transmitted disease (STD) clinics in the United States served as recruitment sites. Sensitivity and specificity estimates, head-to-head comparisons, measures of agreement, and latent-class analyses were performed to provide robust estimates of performance. A total of 508 participants (174 men and 334 women) with anogenital lesions were included; 260 HSV-2 and 73 HSV-1 infections were identified. No differences in test performance based on gender, clinic type, location of the lesion, or type of lesion were observed. The sensitivity of HSV-2 detection ranged from 98.4 to 100% depending on the analytical approach, while the specificity ranged from 80.6%, compared to the less sensitive culture method, to 97.0%, compared to PCR. For HSV-1, the sensitivity and specificity ranges were 96.7 to 100% and 95.1 to 99.4%, respectively. This assay may improve our ability to accurately diagnose anogenital lesions due to herpes infection.     

Zinc salts inactivate clinical isolates of herpes simplex virus in vitro

Using a standard plaque assay and clinical isolates of herpes simplex virus (HSV), we have tested the ability of zinc salts to inactivate HSV. Virus was treated by incubation at 37 degrees C with zinc salts in morpholinepropanesulfonic acid-buffered culture medium and was then diluted and plated onto CV-1 cells for detection and quantitation of remaining infectious virus. Of 10 randomly chosen clinical isolates (five HSV type 1 [HSV-1] isolates and five HSV-2 isolates), seven were inactivated >98% by treatment in vitro with 50 mM zinc gluconate for 2 h and nine were inactivated >97% by treatment with zinc lactate. The effect was concentration dependent. With an HSV-1 isolate, 50 mM zinc gluconate or zinc lactate caused 100% inactivation, 15 mM caused 98 to 99% inactivation, and 5 mM caused 63 to 86% inactivation. With an HSV-2 isolate, 50 and 15 mM zinc gluconate caused 30% inactivation and 5 and 1 mM caused less than 9% inactivation, whereas 50 and 15 mM zinc lactate caused greater than 92% inactivation and 5 and 1 mM caused 37 and 26% inactivation, respectively. The ability of the zinc salts to inactivate HSV was not related to pH in the pH range of 6.1 to 7.6 since inactivation by zinc gluconate or zinc lactate in that pH range was 99.7 to 100% with a 2-h treatment with 50 mM zinc salt. Short (5-min) treatments of selected isolates with zinc gluconate, zinc lactate, zinc acetate, or zinc sulfate yielded inactivation rates of 0 to 55%.     

Evaluation of three glycoprotein G2-based enzyme immunoassays for detection of antibodies to herpes simplex virus type 2 in human sera

Three new glycoprotein G-based enzyme immunoassays (ETI-HSVK-G 2, Sorin Diagnostics Biomedica [assay A]; HSV Type 2 Specific IgG ELISA, Gull Laboratories, Inc. [assay B]; Cobas Core HSV-2 IgG EIA, Roche [assay C]) for the detection of herpes simplex virus (HSV) type 2 (HSV-2)-specific antibodies were evaluated. By testing sera from 25 individuals with culture-proven HSV-2 infection, the assays showed a sensitivity of 96%. The specificities, evaluated with sera from 70 HSV antibody-negative children, 75 HSV antibody-positive children, and 69 HSV antibody-negative adults, were 100% for assay A, 96.2% for assay B, and 97.8% for assay C, respectively. Discrepant results by any of the three assays, i.e., reactivity of a specimen in only one or two assays, occurred with similar frequencies for HSV-seronegative individuals as well as HSV-seropositive children and adults. For sera with discrepant results, the positive reactivity was mostly low. Thus, for determination of the prevalence of HSV-2 antibodies, only concordantly positive results were considered. On the basis of the results obtained with sera from 41 adults with culture-proven HSV-1 infection and from 173 HSV-antibody-positive pregnant women, the HSV-2 seroprevalence was 9. 8%. The results show that the new glycoprotein G2-based enzyme immunoassays are useful tools for the detection of type-specific HSV-2 antibodies. However, if only one assay is performed, careful interpretation of the results is indicated, especially if the exhibited reactivity is low, and for determination of the definitive HSV-2 serostatus, confirmatory assays may still be necessary.     

A MOLECULAR METHOD FOR TYPING HERPES SIMPLEX VIRUS ISOLATES AS AN ALTERNATIVE TO IMMUNOFLUORESCENCE METHODS

Background: Typing of Herpes simplex virus (HSV) isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF) assays and polymerase chain reaction (PCR). This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb) based IF test. Study design: This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase (pol) gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42) of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42) were HSV-2, and two (4.8%) were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30), whereas the cost per MAb IF test is about Rs 1,500 (USD 35) including all overheads (reagents, instruments, personnel time, and consumables). Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers) is now more widespread than fluorescence microscopes in a country like India.