Limits in Reliability of Glycoprotein G-Based Type-Specific Serologic Assays for Herpes Simplex Virus Types 1 and 2

Type-specific serologic assays for herpes simplex virus (HSV) types 1 and 2 based on glycoprotein G-1 (gG-1) (HSV-1) and gG-2 (HSV-2) discriminate between antibodies against HSV-1 and HSV-2. We previously developed a Western blot assay using gG-1 and gG-2 expressed in baculovirus, performed extensive validation studies, and determined that it was both sensitive and specific for type-specific detection of HSV antibody. Here we report that, among a cohort of Thai military recruits, the serostatus of some individuals changed from positive to negative over time (6.6% among those ever positive for HSV-1, and 14.9% among those ever positive for HSV-2). We tested a subset of these specimens in three other gG-based assays: an enzyme-linked immunosorbent assay, an immunoblot strip assay, and a Western blot assay. Positive-to-negative shifts occurred in every assay; the frequency of the shifts ranged from 6.1% to 21.2% of the specimen sets tested. There was only limited agreement among the assays concerning which individuals lost reactivity. This inaccuracy, exhibited by all of the assay protocols, was not predicted by validation studies employing specimens from cross-sectional studies and was most pronounced in HSV-2 testing. This argues for the inclusion of serial blood specimens in serologic assay validation procedures.     

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.     

Novel Approach for Specific Detection of Herpes Simplex Virus Type 1 and 2 Antibodies and Immunoglobulin G and M Antibodies

Recently a few new herpes simplex virus (HSV) type-specific serological diagnostic tests have been introduced to the commercial market, but these tests have some limitations. Moreover, it is not yet clear which commercial test can be regarded as a “gold standard” for the serodiagnosis of HSV infections. In order to improve the clinical diagnostic value of serological tests for the detection of HSV infections, we developed novel, competition-based enzyme-linked immunosorbent assays for the specific determination of HSV type 2 antibodies (SeroHSV2) and HSV type 1 antibodies (SeroHSV1) and two complementary tests for the detection of HSV immunoglobulin M (IgM) and IgG antibodies (SeroHSV IgM and SeroHSV IgG). These four new kits were evaluated in comparison with some commercial kits for the detection of HSV antibodies that are commonly used at present in Israeli clinical laboratories. The results indicate that SeroHSV2 is highly sensitive (>92%) and highly specific (>94%). SeroHSV2 does not cross-react with other alphaherpesvirus antibodies. SeroHSV1 is highly sensitive (>94%) and specific (>91%) compared to four commercial available kits. SeroHSV IgM is highly specific (>92%) in comparison with other commercial HSV IgM tests. The sensitivity of SeroHSV IgM ranges between 50 and 70% compared to these tests. Further investigation of the discrepant results obtained by using in-house competition tests indicated that SeroHSV IgM is more sensitive. SeroHSV IgG was also found to be highly sensitive (>94%) and highly specific (>92%) compared to the other commercial HSV IgG tests.     

Evaluation of Three Multiplex Flow Immunoassays Compared to an Enzyme Immunoassay for the Detection and Differentiation of IgG Class Antibodies to Herpes Simplex Virus Types 1 and 2

The diagnosis of herpes simplex virus (HSV) infections is routinely made based on clinical findings and supported by laboratory testing using PCR or viral culture. However, in instances of subclinical or unrecognized HSV infection, serologic testing for IgG class antibodies to type-specific HSV glycoprotein G (gG) may be useful. This study evaluated and compared the performances of three multiplex flow immunoassays (AtheNA Multi-Lyte [Zeus Scientific], BioPlex 2200 [Bio-Rad Laboratories], and Plexus HerpeSelect [Focus Diagnostics]) for the simultaneous detection of gG type-specific IgG antibodies to HSV types 1 and 2 (HSV-1 and HSV-2). Serum specimens (n = 505) submitted for routine gG type-specific HSV IgG testing by enzyme immunoassay (EIA) (HerpeSelect; Focus Diagnostics) were also tested by the three multiplex flow immunoassays. Specimens showing discordant results were tested by HSV type-specific Western blotting (WB). For HSV-1 IgG, the AtheNA, BioPlex, and Plexus assays demonstrated agreements of 94.9% (479/505 specimens), 97.8% (494/505 specimens), and 97.4% (492/505 specimens), respectively, with the results of EIA. For HSV-2 IgG, the AtheNA, BioPlex, and Plexus assays showed agreements of 87.9% (444/505 specimens), 97.2% (491/505 specimens), and 96.8% (489/505 specimens), respectively, with EIA results. Timing studies showed that the AtheNA, BioPlex, and Plexus assays could provide complete analysis of 90 serum specimens in 3.1, 1.5, and 2.9 h, respectively, versus 3.1 h by EIA. These findings suggest that the gG type-specific HSV IgG multiplex immunoassays may be beneficial to high-volume clinical laboratories experiencing significant increases in the number of specimens submitted for HSV serologic testing. The evaluated systems provide comparable results to those of EIA, while reducing hands-on time and eliminating the necessity to aliquot specimens prior to testing.Herpes simplex virus type 1 (HSV-1) and HSV-2 are common causes of disease worldwide, with transmission resulting from direct contact with virus-infected secretions. The prevalence of HSV-1 infection increases with age, and >70% of adults worldwide are seropositive for the virus (18). The incidence of antibodies to HSV-2 is dependent on age, sex, and risk factors (e.g., number of sexual partners) and may reach 60 to 95% in certain high-risk groups, such as patients infected with HIV (11, 13, 20). However, a relatively small percentage of patients (10 to 20%) know that they are infected with genital herpes (12, 13, 21), thereby contributing to increased transmission of disease.The diagnosis of HSV-associated disease is routinely made based on clinical findings and supported by laboratory testing using PCR or viral culture (13, 22). However, in instances of subclinical or unrecognized HSV infection, serologic testing for immunoglobulin G (IgG) antibodies to type-specific HSV glycoprotein G (gG) may be useful. Due to significant antigenic cross-reactivity among HSV structural proteins, only gG-based serologic assays have been shown to accurately differentiate between IgG class antibodies to HSV-1 and HSV-2 (2, 4, 9, 15), and FDA-approved, conventional, HSV type-specific enzyme immunoassays (EIA) and Western blot (WB) assays are commercially available. Although EIA and WB have demonstrated excellent sensitivity and specificity (2, 3, 15, 24), they require separate assays to be performed for the detection and differentiation of antibodies to HSV-1 and HSV-2. This may increase the potential for aliquoting errors, as well as the associated technologist and instrument time required for testing.Recently, a number of multiplex flow immunoassays (MFI) have been described for the serologic evaluation of various infectious diseases (6, 8, 16). This approach is similar to traditional EIA but allows for the simultaneous detection and identification of multiple analytes in a single reaction tube. MFI technology uses a liquid suspension array of up to 100 unique microspheres (5- to 6-μm beads), each conjugated to a different capture molecule (e.g., antibody, antigen, or nucleic acid). Each capture analyte is detected and quantitated following the addition of a fluorescently labeled reporter molecule (e.g., phycoerythrin) whose emission is measured by a flow-based detector. Since 2008, three multiplex flow immunoassays (AtheNA Multi-Lyte [Zeus Scientific], BioPlex 2200 [Bio-Rad Laboratories], and Plexus HerpeSelect [Focus Diagnostics]) have received FDA clearance for the detection and differentiation of IgG class antibodies to HSV-1 and HSV-2. These assays are fully automated and designed for high-throughput analysis of the HSV type-specific antibody response.Due to increasing test volumes (∼105% in the past 3 years) and the limitations of conventional methods for HSV antibody testing (e.g., limited throughput and labor-intensive testing), we undertook a study to evaluate and compare the AtheNA, BioPlex, and Plexus multiplex assays for the detection and differentiation of IgG class antibodies to HSV-1 and HSV-2. The objective of this study was to compare the results of MFI and EIA testing, using WB to further evaluate specimens showing discordant results.     

Comparison of Chemicon SimulFluor Direct Fluorescent Antibody Staining with Cell Culture and Shell Vial Direct Immunoperoxidase Staining for Detection of Herpes Simplex Virus and with Cytospin Direct Immunofluorescence Staining for Detection of Varicella-Zoster Virus

A new rapid direct immunofluorescence assay, the SimulFluor direct fluorescent-antibody (DFA) assay, which can simultaneously detect herpes simplex virus types 1 and 2 (HSV-1 and -2) and varicella-zoster virus (VZV), was evaluated in comparison with our current standard procedures of (i) shell vial direct immunoperoxidase (shell vial IP) staining and cell culture for detection of HSV and (ii) cytospin DFA staining for VZV detection. A total of 517 vesicular, oral, genital, and skin lesion specimens were tested by all three procedures. For HSV detection, the SimulFluor DFA assay had an overall sensitivity, specificity, positive predictive value, and negative predictive value of 80.0, 98.3, 92.3, and 95.1%, respectively, when compared to culture. Shell vial IP staining had a sensitivity, specificity, positive predictive value, and negative predictive value of 87.6, 100, 100, and 96.9%, respectively, when compared with cell culture. The SimulFluor DFA assay, however, offers same-day, 1.5-hours results versus a 1- to 2-day wait for shell vial IP staining results and a 1- to 6-day wait for culture results for HSV. For VZV detection SimulFluor DFA staining detected 27 positive specimens as compared to 31 by our standard cytospin DFA technique—a correlation of 87.1%. A positive SimulFluor reaction for VZV is indicated by yellow-gold fluorescence compared to the bright apple-green fluorescence observed by cytospin DFA staining. There is no difference in turnaround time between the two assays. The SimulFluor DFA assay is a rapid immunofluorescence assay that can detect 80% of the HSV-positive specimens and 87% of the VZV-positive specimens with a 1.5-h turnaround time.     

Analysis of varicella-zoster virus and herpes simplex virus in various clinical samples by the use of different PCR assays

Rapid and reliable detection of varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and -2 (HSV-2) is of clinical significance in immunocompromised patients and patients with infections of the central nervous system. This paper describes the detection of VZV and HSV using the commercially available Affigene^® VZV and Affigene^® HSV 1/2 tracer kits in comparison to “in-house” polymerase chain reaction (PCR) assays. For sample preparation, Qiagen (Hilden, Germany) and Affigene^® (Cepheid AB, Bromma, Sweden) DNA extraction kits were used. 175 samples were analyzed for VZV and 352 samples for HSV-1 and -2. Generally more positive results were obtained using the Affigene^® assays compared to the “in-house” methods independent of the DNA preparation method used. There were significant differences in sensitivity between the Affigene^® HSV 1/2 tracer and the “in-house” PCR assays for the detection of both HSV-1 and -2 in cerebrospinal fluid and vesicle/skin swabs. The Affigene^® HSV 1/2 and VZV tracers are very sensitive assays for detection of VZV and HSV. A wide variety of clinical samples can be examined in combination with either the Qiagen or the Affigene^® DNA extraction kits for preparation.     

Effect of sexually transmitted disease (STD) coinfections on performance of three commercially available immunosorbent assays used for detection of herpes simplex virus type 2-specific antibody in men attending Baltimore, Maryland, STD clinics

Two hundred seventy-nine serum samples from men attending sexually transmitted disease (STD) clinics in Baltimore, Maryland, were tested for herpes simplex virus type 2 (HSV-2)-specific antibody by three immunosorbent glycoprotein G-2-based assays (the Kalon, Focus, and Biokit assays). The results for all samples with positive results were confirmed by Western blotting (91/279; 32.6% HSV-2 seroprevalence). All patients were also tested for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, human immunodeficiency virus type 1, and hepatitis C virus. The Kalon assay performed very well with samples from this population (90.8% sensitive, 99.4% specific), whereas the Focus assay had a sensitivity (82.6%) much lower than that shown previously. For 19.7% of the samples, the Biokit assay gave an indeterminate result. It was found that the odds of a sample having a Biokit assay indeterminate result compared to that of having a definitive positive or negative results were 3.88 times greater for subjects concurrently infected with N. gonorrhoeae, after the effects of other STDs were controlled for (P = 0.001; 95% confidence interval, 1.78, 8.45). Unfortunately, we were unable to control for HSV-1 infection status in the regression model, which, on the basis of χ² analysis, might also affect the clarity of the Biokit test. The recommended index cutoff value of 1.1 for the Focus and Kalon assays was found to be optimal for this population.     

Comparison of Two Enzyme-Linked Immunosorbent Assays and One Rapid Immunoblot Assay for Detection of Herpes Simplex Virus Type 2-Specific Antibodies in Serum

The sensitivities and specificities of three immunoassays for the detection of herpes simplex virus type 2 (HSV-2)-specific immunoglobulin G antibodies in serum, including the one-strip rapid immunoblot assay (RIBA; Chiron Corporation) and two indirect enzyme immunosorbent assays (EIA; Gull Laboratories and Centocor), were compared by testing a panel of 1,250 serum samples from individuals attending an outpatient clinic for sexually transmitted diseases. A qualitative agreement among the three assays was observed with 1,080 serum samples (86.4%); 291 of the serum samples (23.3%) were positive, 789 samples (63.1%) were negative, and 170 serum samples (13.6%) gave a discordant result. Results were considered conclusive when a concordant result was obtained with two of three assays. The sensitivities and specificities of the RIBA, the Gull EIA, and the Centocor EIA proved to be 99.2, 99.7, and 89.9% and 97.1, 96.7, and 99.3%, respectively. These results indicate that the Chiron RIBA and the Gull EIA are especially useful and reliable for the detection of HSV-2-specific antibodies in serum.     

Detection and Direct Typing of Herpes Simplex Virus in Perianal Ulcers of Patients with AIDS by PCR

The presence of herpes simplex virus type 1 (HSV-1) and HSV-2 in perianal ulcerations of 41 AIDS patients was assessed by virus culture and a type-specific PCR-based assay. HSV was isolated from the lesion site in 24 of 41 (58.5%) patients, and HSV DNA was detected by PCR in all 24 (100%) of these specimens. Additionally, PCR was used to detect HSV DNA in 12 of 17 (70.5%) HSV culture-negative samples. Thus, HSV genomic sequences could be demonstrated in 36 of 41 (87.8%) perianal ulcers in this series. Full agreement in HSV typing by either immunodot assay or PCR was seen in 24 samples that were positive by both virus culture and PCR. HSV-2 was demonstrated in 35 of 36 (97.2%) HSV-positive samples.     

Comparative Performance of a Novel Herpes Simplex Virus Type 2-Specific Enzyme-Linked Immunosorbent Assay Using a Targeted Chain Oligopeptide,Peptide 55

Herpes simplex virus (HSV) glycoprotein G (gG2) has been used as the basis of many serological assays for the detection of HSV type 2 (HSV-2)-specific antibodies. In the present study, an enzyme-linked immunosorbent assay (ELISA), the Pathozyme Viro HSV-2 immunoglobulin G (IgG) ELISA (Omega Diagnostics, Alva, United Kingdom), based on an immunodominant epitope of gG2 presented in a branched-chain format (peptide 55), was compared with two commercially available gG2-specific assays, the Bioelisa HSV-2 IgG assay (Biokit, S.A., Barcelona, Spain) and the HerpesSelect HSV-2 IgG assay (Focus Diagnostics, Cypress, CA). A panel of 218 well-characterized serum samples was tested. Thirty-one samples were determined to be HSV-2 IgG antibody positive and 164 samples were determined to be negative with all three kits. The levels of concordance between the tests were 95.9% between the Omega and HerpeSelect assays, 90.8% between the Omega and Bioelisa assays, and 94.5% between the HerpeSelect and Bioelisa assays. Twenty-three samples gave discordant results. Western blot results showed that of these, the results for 77% were correctly identified by the Omega assay, the results for 68% were correctly identified by the HerpeSelect assay, and the results for 13.6% were correctly identified by the Bioelisa assay. Although there was a high level of agreement between the results obtained by the three assays and no false-positive results were detected by any of the three kits, confirmation of the results for samples with discordant results by Western blotting suggested that the peptide 55-based Omega assay is the most sensitive and specific assay among the assays evaluated.Genital herpes is one of the most common sexually transmitted infections worldwide. Historically, herpes simplex virus type 2 (HSV-2) has predominantly been associated with genital infections; however, recent reports suggest that a considerable and increasing number of genital isolates are of HSV-1 (10, 24). The clinical course of primary genital herpes infections among patients infected with HSV-1 and HSV-2 are similar; however, there are differences in epidemiology and natural history of the diseases caused by the two viral subtypes (9). HSV-2 primary infection during pregnancy has been related to spontaneous abortion, prematurity, congenital infection, and neonatal herpes. Several recent studies have reported that genital herpes may increase the susceptibility of acquiring human immunodeficiency virus by persons with high-risk behavior, because HSV-2 can cause breaks in the genital mucosal barrier (16).HSV-1 and HSV-2 have approximately 83% nucleotide sequence similarity, and for some proteins they share more than 85% identity (4). Both serotypes therefore show extensive serological cross-reactivity. This has impeded seroepidemiologic studies of the two viruses. The major antigenic determinants are glycoprotein antigens exposed on the virion surface (22), and although HSV-1 and HSV-2 are known to express more than 11 glycoproteins, 10 of these glycoproteins express multiple epitopes common to each type. A variety of studies have concluded that only glycoprotein G (gG2) expresses epitopes specific for HSV-2 (14, 21). Consequently, this glycoprotein has been used as the basis of type-specific serological assays for the diagnosis of HSV-2 infection (1, 2, 7, 19, 20). However, epitope mapping of gG2 by Levi et al. (11) suggested that some epitopes may cross-react with HSV-1-specific antibody.In an effort to develop even greater selectivity for HSV-2, several research groups have studied HSV-2 type-specific epitopes within gG2. Regions comprising amino acids 350 to 427 and 525 to 587 and also regions comprising amino acids 625 to 641 and 676 to 699 were identified by two independent groups (6, 11), and a secreted protein of gG2 comprising amino acids 23 to 340 was described by Liljeqvist et al. (13) and Gorander et al. (5). Marsden et al. (15) developed multiple antigenic peptides corresponding to residues 561 to 578 of gG2, designated peptide 55. This peptide comprises four peptide copies attached to a branched lysine core and separated from the lysine core by four glycine residues. The sensitivity of the peptide for the detection of HSV-2 antibody was evaluated by screening a panel of HSV-2-positive human serum samples previously characterized by virus isolation and typed by indirect immunofluorescence with a type-specific monoclonal antibody. Peptide 55 was found to be sensitive and specific, and no false-positive results were detected with HSV-1 antibody-positive or HSV-1 or HSV-2 antibody-negative samples.Oladepo et al. (18) compared the performance of an assay with peptide 55 with that of a commercially available HSV-2-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) kit based on affinity-purified gG2 (Gull Laboratories) and found that peptide 55 had the same sensitivity; however, it was more specific than the complete protein. Nilsen et al. (17) tested the performance of an ELISA based on peptide 55 and compared it with the performance of two different assays, an ELISA developed by Ho et al. (8) based upon native gG2 selected by affinity to Helix pomatia lectin, and an ELISA based on affinity-purified gG2 (Gull Laboratories), and found that the performance of the assay with peptide 55 was better than that of the other ELISA methods.In the present study, an ELISA (Pathozyme Viro HSV-2 ELISA IgG; Omega Diagnostics Ltd., Scotland) based on peptide 55 was compared with two other commercially available HSV-2-specific IgG ELISAs, the HerpeSelect HSV-2 ELISA (Focus Diagnostics, Cypress, CA), which uses purified recombinant type-specific gG2 antigen, and the Bioelisa HSV-2 IgG-specific assay (Biokit, S.A., Barcelona, Spain), which uses affinity-purified gG2 protein, for the abilities to detect and serologically differentiate HSV-2 antibody. The three assays are referred to in this study as the HerpeSelect, Bioelisa, and Omega assays, respectively.     

Rapid Screening Tests for Determining In Vitro Susceptibility of Herpes Simplex Virus Clinical Isolates

The susceptibility of human herpes simplex virus (HSV) to acyclovir (ACV) was determined with the use of a single dose of the drug (1 and 2 μg of ACV per ml for HSV-1 and HSV-2, respectively) in two rapid assays: a rapid cytopathic effect inhibitory assay (Rapid CIA) and a rapid dye uptake assay (Rapid DUA). These tests allow the simultaneous determination of virus titer and susceptibility to ACV at a determined viral concentration (100 50% tissue culture infective doses and 100 50% dye uptake units). These tests were compared with a conventional susceptibility assay (dye uptake assay) and showed similar results. Indeterminate results with the Rapid CIA appeared in 3 of 30 samples. With the use of both Rapid CIA and Rapid DUA, we were able to determine the susceptibility of 100% of the isolates. The rapid tests, unlike conventional assays, are able to provide susceptibility results within 3 days after the virus has been isolated from a clinical specimen and could thus play a direct role in therapeutic decisions.     

Detection of a molecular biomarker for zygomycetes by quantitative PCR assays of plasma, bronchoalveolar lavage, and lung tissue in a rabbit model of experimental pulmonary zygomycosis

We developed two real-time quantitative PCR (qPCR) assays, targeting the 28S rRNA gene, for the diagnosis of zygomycosis caused by the most common, clinically significant Zygomycetes. The amplicons of the first qPCR assay (qPCR-1) from Rhizopus, Mucor, and Rhizomucor species were distinguished through melt curve analysis. The second qPCR assay (qPCR-2) detected Cunninghamella species using a different primer/probe set. For both assays, the analytic sensitivity for the detection of hyphal elements from germinating sporangiospores in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates from rabbits was 1 to 10 sporangiospores/ml. Four unique and clinically applicable models of invasive pulmonary zygomycosis served as surrogates of human infections, facilitating the validation of these assays for potential diagnostic utility. For qPCR-1, 5 of 98 infarcted lung specimens were positive by qPCR and negative by quantitative culture (qCx). None were qCx positive only. Among 23 BAL fluid samples, all were positive by qPCR, while 22 were positive by qCx. qPCR-1 detected Rhizopus and Mucor DNA in 20 (39%) of 51 serial plasma samples as early as day 1 postinoculation. Similar properties were observed for qPCR-2, which showed greater sensitivity than qCx for BAL fluid (100% versus 67%; P = 0.04; n = 15). The assay detected Cunninghamella DNA in 18 (58%) of 31 serial plasma samples as early as day 1 postinoculation. These qPCR assays are sensitive and specific for the detection of Rhizopus, Mucor, Rhizomucor, and Cunninghamella species and can be used for the study and detection of infections caused by these life-threatening pathogens.     

Laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex PCR screening assay

A multiplex PCR assay that detects the four commonest causes of viral meningitis and encephalitis in the United Kingdom (herpes simplex virus [HSV] type 1 [HSV-1], HSV type 2 [HSV-2], varicella-zoster virus [VZV], and enteroviruses) was developed, and its sensitivity was compared with those of similar assays described previously for this application. Compared to the previous assays, this single multiplex PCR assay had higher molecular sensitivities for the detection for each of the viruses and improved utility for routine use in a diagnostic laboratory. The assay was used to test a series of 1,683 consecutive cerebrospinal fluid (CSF) samples between June 1997 and March 1998 inclusively. Viral nucleic acid was detected in 138 (8.2%) of the CSF samples, including enteroviruses in 51 samples, HSV-2 in 33 samples, VZV in 28 samples, and HSV-1 in 25 samples. Compared to the accepted relative incidence of viral etiologies, aseptic meningitis due to HSV-2 infection was high, and in adult female patients with symptoms of aseptic meningitis, HSV-2 was the virus most commonly detected in the CSF.     

Rapid, sensitive, and specific lateral-flow immunochromatographic point-of-care device for detection of herpes simplex virus type 2-specific immunoglobulin G antibodies in serum and whole blood

Herpes simplex virus type 2 (HSV-2) is a common human pathogen that can cause a variety of clinical manifestations in humans. In order to provide near-patient results to allow for faster counseling and treatment, a rapid point-of-care test that is accurate and simple to use is desirable. Here, we describe the development and evaluation of an HSV-2 immunoglobulin G (IgG)-specific antibody lateral-flow immunochromatographic assay (LFIA) based on colloidal gold nanoparticles. A total of 359 serum samples and 100 whole-blood samples were tested in the newly developed HSV-2 LFIA. Serum results were compared to those from the HerpeSelect HSV-2 enzyme-linked immunosorbent assay (ELISA), and whole-blood sample results were compared to those of both ELISA and HerpeSelect HSV-1 and -2 immunoblotting (IB). The sensitivity of the HSV-2 LFIA compared to that of the HerpeSelect ELISA was 100% (89/89), and the specificity was 97.3% (257/264). Cross-reactivity with HSV-1 IgG-positive serum samples was observed in 2.6% (5/196) of samples, 2.9% (1/34) for rubella virus, and 6.2% (1/16) for Epstein-Barr virus. No cross-reactivity in varicella-zoster virus or cytomegalovirus IgG-positive serum samples was observed. No interference was observed from bilirubin-, triglyceride-, albumin-, or hemoglobin-spiked samples. The concordance of the LFIA results between capillary whole blood, EDTA-treated venous whole blood, heparin-treated venous whole blood, and serum was 99% (99/100). In conclusion, the LFIA for HSV-2 IgG-specific antibodies demonstrated excellent sensitivity, specificity, and concordance for both serum and whole-blood samples compared to the sensitivity, specificity, and concordance of both HSV-2 ELISA and IB.     

Premarket Evaluation of a Commercial Glycoprotein G-Based Enzyme Immunoassay for Herpes Simplex Virus Type-Specific Antibodies

A new commercial glycoprotein G-based enzyme immunoassay (gG-EIA) was compared with Western blotting (WB) for detection of herpes simplex virus type 1 (HSV-1) or HSV-2 type-specific antibodies in 193 serum samples. Sensitivity for HSV-1 was 95%; specificity was 96%. Sensitivity for HSV-2 was 98%; specificity was 97%. Twelve of 13 serum samples with equivocal gG-EIA results were serotyped by WB.     

Comparison of conventional, nested, and real-time PCR assays for rapid and accurate detection of Vibrio vulnificus

We conducted a prospective study to target toxR in the blood of patients with skin and soft tissue infections who were admitted to four tertiary hospitals to assess the clinical usefulness of real-time quantitative PCR (Q-PCR) as a diagnostic technique. We performed conventional PCR (C-PCR), nested PCR (N-PCR), and Q-PCR assays and compared the results to those obtained using the “gold standard” of microbiological culture. The lower detection limit for the Q-PCR assay was 5 × 10⁰ copies/μl. By use of blood samples of patients with skin and soft tissue infections, the sensitivities of the C-PCR and N-PCR assays against the target toxR gene of V. vulnificus as diagnostic tools were determined to be 45% and 86%, respectively. The C-PCR and N-PCR assays had specificities of 100% and 73%, respectively. When we adopted a crossing-point (cp) cutoff value of <38 cp as a positive result, the Q-PCR assay had 100% sensitivity and specificity. Q-PCR to detect V. vulnificus-specific genes is not only the most sensitive and specific of the techniques but also the most rapid diagnostic method. Therefore, the appropriate application of the Q-PCR assay using blood is useful for the rapid diagnosis and subsequent treatment of V. vulnificus sepsis.     

Comparison of indirect hemagglutination and51Chromium release tests for detection of herpes simplex virus types 1 and 2 antibodies in patients with recurrent herpes infections

Summary Indirect hemagglutination and⁵¹Cr release tests (IHAT and 51-CRT respectively) were compared in patients with recurrent herpes simplex virus (HSV) infections from whom HSV-1 or HSV-2 was isolated. Both tests were equally sensitive and specific in detecting HSV antibodies. However, IHAT was more specific in detecting homologous HSV antibody response in patients with recurrent HSV-2 infections. Past infections with HSV-1 in the patients with dual infections were detected by determining HSV-type specific antibodies by inhibition of IHAT. Cross absorption studies showed that the antibody reactivity measured by the two tests was qualitatively and quantitatively different. Nevertheless, IHAT has been found to be more appropriate test for seroepidemiologic studies of HSV-2 infections because of its specificity, rapidity and less cost, whereas, 51-CRT appears to measure antibodies against recent and more predominant type of infecting HSV.     

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.     

Detection of herpes simplex virus type 2-specific antibody with glycoprotein G.

A recently described herpes simplex virus (HSV) type 2 (HSV-2)-specific glycoprotein (gG-2) was purified on an immunoaffinity column prepared with monoclonal antibody. This purified antigen was used in an immunodot enzymatic assay on nitrocellulose paper for the detection of HSV-2 antibodies in human serum. The test was very sensitive in that HSV-2 antibodies were detected in the convalescent sera of 132 of 134 patients with recurrent genital infections in which HSV-2 had been isolated earlier. Antibodies to gG-2 were detected in 17% of sera obtained within 10 days after the onset of a primary HSV infection and in 95% of sera obtained more than 10 days after onset. The specificity of the immunodot assay was demonstrated by testing sera from 245 HSV-seronegative adults, 344 children, 29 nuns, and 13 patients with primary genital HSV-1 infections. None of these 631 sera was reactive with the gG-2 antigen. When compared with a microneutralization test, the immunodot assay was found to be more specific in detecting HSV-2 antibodies. Reproducibility of the gG-2 assay, obtained by retesting 391 sera, was 95%. Thus, this assay has the sensitivity, specificity, and reproducibility necessary for the measurement of HSV-2 antibodies in seroepidemiological studies.     

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.