Indirect ELISA for the detection of HSV-2 specific IgG and IgM antibodies with glycoprotein G (gG-2).

The glycoprotein G (gG-2) purified from HSV-2 infected cells has been reported to be useful for determination of HSV-2 type-specific antibodies using conventional ELISA formats. This study further confirmed the specificity of gG-2 and demonstrated the feasibility of a specific IgM assay. The gG-2 ELISA was developed to detect HSV-2 specific IgG and IgM antibodies in human sera with high levels of sensitivity and specificity. Of 45 patients with culture-proven recurrent HSV-2 genital infection 44 were reactive for gG-2 IgG. Of 30 sera from patients with culture-proven recent initial HSV-2 genital infection 29 were positive for gG-2 IgM. Three patients with primary HSV-2 genital infection showed gG-2 IgM in the convalescent but not in the acute sera. The IgG- and IgM-gG-2 ELISA showed high specificity. None of 40 sera from children were reactive by either assay. Only one of 94 sera from patients with antibody to herpesviruses other than HSV reacted in the IgG assay but none reacted in the IgM assay. There was no cross-reaction with sera from patients with proven HSV-1 infection with the gG-2 antigen. The results suggest that the IgG assay can be used for demonstration of past HSV-2 infection and the IgM assay for the diagnosis of HSV-2 in neonatal herpes and primary genital herpes, when cultures or rapid diagnostic techniques are unavailable.     

Detection of immunoglobulin M antibodies to glycoprotein G-2 by western blot (immunoblot) for diagnosis of initial herpes simplex virus type 2 genital infections.

Western blots (immunoblots) for the detection of immunoglobulin M (IgM) antibodies specific for herpes simplex virus type 1 (HSV-1) and HSV-2 in patients' sera were developed. The locations of the type-specific glycoprotein G (gpG-2) of HSV-2 (92- and 140-kDa forms) and glycoprotein C of HSV-1 (gpC-1), which carries mostly type-specific antigenic epitopes, were checked with specific monoclonal antibodies. Western blot assays for IgM antibody to gpC-1 or gpG-2 were performed after depletion of IgG by precipitation with anti-human IgG. In patients with primary HSV-2 genital infections, seroconversion of IgM and IgG antibodies to both the 92- and 140-kDa forms of gpG-2 was observed, although both antibodies appeared in convalescent-phase serum after the first week. IgM and IgG antibodies to low-molecular-size polypeptides (40 to 65 kDa) were the first antibodies observed in patients with primary infection, but these antibodies were cross-reactive with HSV-1 and HSV-2. However, in patients with recurrent HSV-2 infections, IgG antibodies to both forms of gpG-2 and the low-molecular-size polypeptides were found no matter how early after onset the patient was bled, and IgM to gpG-2 did not appear. In patients with nonprimary initial genital HSV-2 infections, IgG antibody to HSV-1 was demonstrated in the first serum specimen, and HSV-2-specific IgM was found in 39% of the serum specimens. Hence, the Western blot assay can be used to test for IgM antibody to gpG-2, allowing for the retrospective diagnosis of inital HSV-2 infections and its use as a supplementary test to the gpG-2 IgG enzyme-linked immunosorbent assays developed elsewhere. In contrast, IgM antibody to gpG-2 is not usually detected in patients with recurrent HSV-2 infections.     

Secreted portion of glycoprotein g of herpes simplex virus type 2 is a novel antigen for type-discriminating serology

The secreted portion of glycoprotein G (sgG-2) of herpes simplex virus type 2 (HSV-2) was evaluated as a novel antigen in an enzyme-linked immunosorbent assay (ELISA) format for detection of type-specific immunoglobulin G (IgG) antibodies in HSV-2-infected patients. The results were compared with those obtained by a commercially available assay, the HerpeSelect 2 ELISA (the FOCUS2 assay). Five different panels of sera were analyzed: panel A consisted of 109 serum samples from patients with a culture-proven HSV-1 infection that were Western blotting (WB) negative for HSV-2; panel B consisted of 106 serum samples from patients with a culture-proven recurrent HSV-2 infection that were WB positive for HSV-2; panel C consisted of 100 serum samples with no detectable IgG antibodies against HSV-1 and HSV-2; panel D consisted of 70 HSV-2 negative "tricky" serum samples containing antinuclear IgG antibodies or IgM antibodies against other viruses or bacteria; and panel E consisted of consecutive serum samples from 21 patients presenting with a first episode of HSV-2-induced lesions. When sera in panels A to C were analyzed, the sgG-2 ELISA and the FOCUS2 assay both showed sensitivities and specificities of >or=98%. In total, among the samples in panel D, 13 serum samples (19%) were false positive by the FOCUS2 assay and 1 serum sample (1.4%) was false positive by the sgG-2 ELISA. When the sera in panel E were analyzed, the sgG-2 ELISA detected seroconversion somewhat later than WB or the FOCUS2 assay did. We conclude that sgG-2 induces an HSV-2 type-specific antibody response and can be used for type-discriminating serology.     

Performance of the BioPlex 2200 Flow Immunoassay in Critical Cases of Serodiagnosis of Toxoplasmosis

The BioPlex 2200 automated analyzer (Bio-Rad Laboratories, Hercules, CA) is a recently developed multiplex analyzer that enables the detection of anti-Toxoplasma, -rubella, and -cytomegalovirus antibodies in the same assay. The aim of this study was to compare this new technology (using the BioPlex 2200 ToRC IgG/IgM kit) in critical cases of serodiagnosis of toxoplasmosis (acute, chronic, or congenital infections and cases with discrepant results) to the technologies used in our routine practice, i.e., the Platelia IgG/IgM enzyme-linked immunosorbent assays (ELISAs) (Bio-Rad Laboratories) and the Toxo-Screen direct agglutination assay (bioMérieux, Lyon, France). Overall, most cases of false-positive/negative results obtained with the Platelia IgG or Toxo-Screen assay were corrected by the BioPlex 2200 ToRC IgG (87.5%). Furthermore, the analysis of 35 sequences of sera showed a trend toward a more rapid decrease of IgM titers by BioPlex 2200 than by Platelia. These results for IgM detection can be explained by a weaker detection of residual IgM. Indeed, among 23 serum samples from patients with probable past infection with long-lasting IgM (Platelia M positive and IgG avidity index, ≥0.5), the BioPlex 2200 Toxoplasma IgM assay was positive for only 11 serum samples. In our panel of critical cases comprising 156 serum and 6 cord blood samples from 103 patients with acute, chronic, or congenital infection, the BioPlex 2200 IgG assay was a sensitive (97.8%) and specific (91.3%) method for IgG detection. The high specificity (97.4%) of IgM detection combined with the shorter kinetics of IgM titers may considerably reduce the number of residual IgM detections, thus yielding more precise diagnoses of acute infections.     

ELISA for detection of IgG and IgM antibodies to HSV-1 and HSV-2 in human sera

A rapid, enzyme-linked immunoassay (ELISA) was applied to identify and measure specific IgG and IgM antibodies to herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2). Detergent solubilized infected cells and mock-infected cells were used as antigens in the assay. Identification of type-specific antibodies was achieved by a competition assay in which clinical sera mixed with HSV-1 or HSV-2 antigens were assayed for reactivity to identical antigens coating wells of polystyrene microtiter plates. Reactivity and the specificity of the reactive immunoglobulin class was quantitated using biotinylated goat anti-IgG and biotinylated goat anti-IgM. Five paired sera from patients with diagnosed herpes simplex genital infections and one human anti-HSV-1 reference serum were tested with this assay and results were compared to results previously obtained using a complement fixation test and micro-SPRIA. The results indicate that the ELISA is a specific, sensitive and simple test which confirms the herpes simplex virus infection history of patients.     

Evaluation of the BioPlex 2200 Syphilis System as a First-Line Method of Reverse-Sequence Screening for Syphilis Diagnosis

Despite recent technological advances, the diagnosis of syphilis remains a challenging enterprise. Actually, most high-volume laboratories have adopted the “reverse algorithm” due several factors, including the potential to automate testing. Recently, immunoassays processed on random-access systems have been proposed as screening tests. The purpose of this study was to evaluate diagnostic performances of BioPlex 2200 Syphilis IgG and BioPlex 2200 Syphilis IgM, tests based on Multiplex Flow technology, in comparison with the performance of Architect Syphilis TP, a chemiluminescent immunoassay for the detection of IgG and/or IgM anti-Treponema pallidum antibodies. A retrospective study was performed with a panel of 100 blood donor sera, a panel of 350 clinical and laboratory-characterized syphilitic sera, and 170 samples obtained from subjects with potentially interfering conditions. Moreover, 200 unselected samples submitted to the Microbiology Laboratory of St. Orsola Hospital in Bologna for routine screening for syphilis were evaluated. As confirmatory tests, T. pallidum hemagglutination and Western blot assays were used. Considering the IgG Western blot (WB) assay to be the gold standard method, BioPlex 2200 Syphilis IgG specificity was far higher than Architect Syphilis TP specificity (89.7% versus 78.4%, respectively), whereas the sensitivity was 100% for both automated methods. Compared to the IgM WB assay, BioPlex 2200 Syphilis IgM performed with a specificity of 94.9%, whereas the sensitivity was 84.8%. Considering the excellent ease of use and automation, the high sample throughput and its valuable analytical performances, BioPlex Syphilis 2200 IgG could represent a suitable choice for high-volume laboratories. BioPlex Syphilis 2200 IgM could be considered a good addition to IgG testing for uncovering active infections.     

Comparison of Western blot (immunoblot) and glycoprotein G-specific immunodot enzyme assay for detecting antibodies to herpes simplex virus types 1 and 2 in human sera.

Sera from patients with culture-proven genital herpes infections were tested for herpes simplex virus type 1 (HSV-1)- and HSV-2-specific antibodies by both a Western blot (immunoblot) technique (WBA) and immunodot enzyme assays (IEAs) specific for HSV-1 or HSV-2 glycoprotein G (gG). Of 137 serum samples tested, none was mistyped by either WBA or IEA. Both tests were most sensitive with sera obtained at least 21 days after onset of primary HSV-2 infections or sera drawn during recurrent HSV-2 genital episodes: 75 of 76 (99%) such serum samples were positive for HSV-2 antibody by WBA and 73 of 76 (96%) were positive by IEA. Of sera drawn earlier than 21 days from onset of primary genital HSV-2, antibodies to HSV-2 were detected in 25% by WBA and 8% by IEA. In patients with culture-proven primary genital HSV-1 infection, WBA detected antibodies to HSV-1 proteins in 16 of 17 (94%) serum samples drawn at least 21 days after onset of primary genital HSV-1 infection, compared with 9 of 17 (53%) serum samples tested for gG-1 by IEA. Both WBA and IEA are accurate and sensitive tests for HSV-2 antibody in patients convalescing from a first episode or having symptomatic or asymptomatic recurrent genital herpes. WBA was more sensitive than IEA in detecting seroconversion following primary HSV-1 genital herpes, although both assays may miss persons undergoing early seroconversion to HSV-2.     

The diagnosis of recent herpes simplex virus type 2 genital infections by the simplex-2 test.

The prevalence of complement-fixing (CF) antibody against the AG-4 early antigen of herpes simplex virus (HSV) type 2 (HSV-2) was determined in patients with culture confirmed HSV-2 genital herpes and control groups using a commercial HSV-2 early antigen (Simplex-2; Gene Link Australia Ltd). Eighty seven per cent of 39 sera collected between 14 and 28 days after confirmed primary and recurrent HSV-2 infection were positive. In acute sera collected between 2-10 days after onset the Simplex-2 test was negative in all 90 patients with presumed primary infection but positive in 53% of 230 sera from recurrent infection. A specificity of 90-94.5% was obtained by testing 36 patients with recent proven HSV-1 infection and 331 control group patients. The Simplex-2 test may be useful in some cases of culture-negative, clinically suspected genital HSV-2 lesions only when sera are collected between 14-28 days after primary and recurrent infection. Its lack of specificity makes it unsuitable for the routine diagnosis of recent HSV-2 infection in the general population.     

Validation of an in-house assay for cytomegalovirus immunoglobulin G (CMV IgG) avidity and relationship of avidity to CMV IgM levels

Measurement of cytomegalovirus (CMV)-specific immunoglobulin G (IgG) avidity has proven to be a powerful tool for distinguishing primary from nonprimary CMV infection. An in-house enzyme-linked immunosorbent assay (ELISA) for measuring CMV IgG avidity was validated using 84 sera from pregnant women who had recently seroconverted following primary CMV infection and 74 sera from individuals with past CMV infection (IgG-positive and IgM-negative profile). Of the 84 sera from pregnant women, 73 sera were collected within 120 days of the last IgG-negative sample, and 72 of these 73 sera (99%) exhibited an avidity index (AI) of <50%. In contrast, 71 of 74 (96%) sera from individuals with past CMV infection exhibited CMV AI values of > 60%. Thus, low avidity in the in-house ELISA was defined as an AI of < or = 50%, whereas high avidity was defined as an AI of > or = 60%. In additional studies, the relationship between CMV IgG avidity and CMV IgM levels was examined using 64 CMV IgG-positive sera (time since seroconversion unknown) exhibiting equivocal or positive results in a CMV IgM capture ELISA (Diamedix). Of these 64 sera, 29 exhibited IgM index values of > or = 3.0, and 27 of these 29 (93%) exhibited low IgG avidity. A similar trend was observed when a subset of these 64 sera (n = 48) was tested in another CMV IgM capture ELISA (Trinity); of 18 sera with IgM index values of > or = 3.0, 17 (94%) exhibited low IgG avidity. These findings demonstrate the validity of an in-house ELISA for CMV IgG avidity and further show that strong reactivity of CMV IgG-positive sera in either of two CMV IgM capture assays is a reliable indicator of low CMV IgG avidity, and thus, recent CMV infection.     

Screening of blood donors for human cytomegalovirus (HCMV) IgG antibody with an enzyme immunoassay using recombinant antigens.

BACKGROUND: Screening of blood donors for human cytomegalovirus (HCMV) infection is usually performed by the combined detection of specific IgG and IgM antibody. However, in most of the cases of primary infection HCMV IgG seroconversion is observed concomitantly to IgM production and HCMV IgM antibody detection for blood donor screening is subject to a relatively high frequency of false positive results. OBJECTIVE: In the present study a newly established HCMV IgG ELISA based on recombinant antigens (anti-HCMV recombinant IgG ELISA, Biotest) was evaluated in terms of sensitivity and specificity for blood donor screening. STUDY DESIGN: A total of 442 serum samples including follow-up sera of five patients suffering from primary HCMV infection, selected seropositive and seronegative blood donors and routine specimens were comparatively investigated with three HCMV antibody ELISAs (anti-HCMV recombinant IgG ELISA, Biotest; Enzygnost anti-CMV/IgG + IgM, Dade Behring; and Captia CMV-TA, Centocor). RESULTS: IgG seroconversion was detected with anti-HCMV recombinant IgG ELISA as early as IgM in all five patients suffering from primary infection. The alternative ELISAs were less sensitive, detecting seroconversion one to three bleeds later in 2 (Enzygnost anti-CMV/IgG + IgM) and 4 patients (Captia CMV-TA), respectively. Anti-HCMV recombinant IgG ELISA showed a 99.1% agreement with Enzygnost anti-CMV/IgG + IgM and/or Western blot in the preselected blood donors and routine specimens. Relatively high numbers of false negative (n=20) and positive results (n=7) were obtained with Captia CMV-TA. CONCLUSIONS: Our preliminary data suggest that HCMV antibody screening of blood donors can be performed reliably by detection of specific IgG provided that a highly sensitive assay system is used.     

Validation of an In-House Assay for Cytomegalovirus Immunoglobulin G (CMV IgG) Avidity and Relationship of Avidity to CMV IgM Levels

Measurement of cytomegalovirus (CMV)-specific immunoglobulin G (IgG) avidity has proven to be a powerful tool for distinguishing primary from nonprimary CMV infection. An in-house enzyme-linked immunosorbent assay (ELISA) for measuring CMV IgG avidity was validated using 84 sera from pregnant women who had recently seroconverted following primary CMV infection and 74 sera from individuals with past CMV infection (IgG-positive and IgM-negative profile). Of the 84 sera from pregnant women, 73 sera were collected within 120 days of the last IgG-negative sample, and 72 of these 73 sera (99%) exhibited an avidity index (AI) of <50%. In contrast, 71 of 74 (96%) sera from individuals with past CMV infection exhibited CMV AI values of >60%. Thus, low avidity in the in-house ELISA was defined as an AI of 50%, whereas high avidity was defined as an AI of 60%. In additional studies, the relationship between CMV IgG avidity and CMV IgM levels was examined using 64 CMV IgG-positive sera (time since seroconversion unknown) exhibiting equivocal or positive results in a CMV IgM capture ELISA (Diamedix). Of these 64 sera, 29 exhibited IgM index values of 3.0, and 27 of these 29 (93%) exhibited low IgG avidity. A similar trend was observed when a subset of these 64 sera (n = 48) was tested in another CMV IgM capture ELISA (Trinity); of 18 sera with IgM index values of 3.0, 17 (94%) exhibited low IgG avidity. These findings demonstrate the validity of an in-house ELISA for CMV IgG avidity and further show that strong reactivity of CMV IgG-positive sera in either of two CMV IgM capture assays is a reliable indicator of low CMV IgG avidity, and thus, recent CMV infection.     

Epstein-Barr virus antibodies in Kawasaki disease

The prevalent ages at onset for Kawasaki Disease (KD) and Epstein-Barr virus (EBV) infection are known to be similar in Korea and Japan. We evaluated the correlation between EBV infection and KD. The antibodies to EBV such as anti-viral capsid antigen (VCA) IgG and IgM, anti-diffuse and restricted early antigen IgG (anti-EADR IgG), and the anti-EBV determined nuclear antigen IgG (anti-EBNA IgG) were examined in 29 KD patients at five separate times sequentially during a period of one year, and also in 14 other children with a past history of KD. The results of each group were compared with those of age-matched controls. The positive rates of anti-VCA IgG and IgM at presentation in the KD patients were 41.4% (12/29) and 0% (0/29), respectively. Only one patient was found to be anti-VCA IgM-positive within two months. There were no cases of anti-VCA IgG except one, anti-EADR IgG and anti-EBNA IgG positive to negative seroconversion during the year. The children with a past history of KD showed higher anti-EBNA IgG-positive rates than the controls (p=0.04). There was no difference in the seropositive rates of the antibodies to EBV, cytomegalovirus, herpes simplex virus and herpes zoster virus. In conclusion, children with KD were noted to have normal immune responses to EBV infection. Children with a past history of KD seemed to be infected with EBV at a later age than children with no history of KD.     

Recombinant truncated nucleocapsid protein as antigen in a novel immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of severe acute respiratory syndrome coronavirus infection

We report the development of an immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) for severe acute respiratory syndrome coronavirus (SARS-CoV) by using recombinant truncated SARS-CoV nucleocapsid protein as the antigen. The newly developed MAC-ELISA had a specificity and sensitivity of 100% as evaluated by using sera from healthy volunteers and patients with laboratory-confirmed SARS. Using serial serum samples collected from SARS patients, the times to seroconversion were determined by IgM antibody detection after SARS-CoV infection. The median time to seroconversion detection was 8 days (range, 5 to 17 days) after disease onset, and the seroconversion rates after the onset of illness were 33% by the first week, 97% by the second week, and 100% by the third week. Compared with the results of our previous report on the detection of IgG, the median seroconversion time by IgM detection was 3 days earlier and the seroconversion rate by the second week after the illness for IgM was significantly higher than by IgG assay. Our results indicating that the IgM response appears earlier than IgG after SARS-CoV infection in consistent with those for other pathogens. Our newly developed MAC-ELISA system offers a new alternative for the confirmation of SARS-CoV infection.     

Class-specific antibody responses to early and late antigens of varicella and herpes simplex viruses

Antibody responses to early antigens of varicella-zoster virus (VZV), simian varicella virus, and herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) were studied in primary, secondary, and latent infections. IgG antibody responses to the early antigens occurred in primary and secondary VZV and HSV infections, and antibodies to early antigens were also demonstrable in healthy individuals with latent VZV and HSV infections, indicating that the presence of antibodies to early antigens cannot be taken as evidence of active infection with the viruses. Patients with current VZV or HSV infections showed heterotypic IgG antibody responses to early antigens of VZV and HSV to the same extent as to late antigens. In all groups of patients, IgG antibody titers to early antigens were similar to those against the corresponding late antigens, and no difference was seen in the reactivity of early antigens produced with four different blocking agents (cytosine arabinoside, bromodeoxyuridine, trisodium phosphonoformate, and cycloheximide). Antibodies of the IgM and IgA classes reacted with both early and late antigens of HSV, but only with late antigens of VZV and simian varicella virus, suggesting that these antibodies may be directed against late proteins that are expressed to a greater extent in HSV-infected cells treated with blocking agents than they are expressed in treated VZV-infected cells. Homologous IgM antibody responses occurred in both primary and secondary VZV infections, but only in primary HSV infections. Heterotypic IgM responses to HSV-2 antigen were noted in a few VZV patients who did not have demonstrable IgG antibody to HSV, suggesting that even in patients without prior experience with HSV, a VZV infection may stimulate the production of IgM antibodies that react with antigens that are shared by VZV and HSV-2. IgA antibodies to late antigens of VZV and HSV were demonstrable in latent, as well as active, infections with these viruses.     

Development and evaluation of an ELISA using secreted recombinant glycoprotein B for determination of IgG antibody to herpes simplex virus.

An ELISA for the determination of IgG antibody to herpes simplex virus (HSV) was developed using a secreted recombinant HSV-1 glycoprotein B (gB-1s) as a solid phase. The clinical validity of the ELISA was established by testing different groups of sera containing HSV-1, HSV-2, or mixed antibody, in parallel with gB-1s ELISA and conventional HSV-1/HSV-2 ELISA. The new gB-1s ELISA detected HSV-1/HSV-2 antibody in sera from 48 subjects with either HSV-1 or HSV-2 past infection as well as in sera from 20 patients with primary infections by either serotype, in complete agreement with the results obtained using conventional ELISA. In 7 patients with HSV-1 encephalitis the kinetics of the gB-1s serum/cerebrospinal fluid antibody-titre ratio paralleled that of conventional ELISA over a period of time of up to 4 years. Acute and convalescent-phase sera from 28 patients with acute infections by human herpesviruses other than HSV did not show a significant cross-reactivity with gB-1s. In conclusion, gB-1s ELISA is a reliable assay for determination of HSV immune status as well as for diagnosis of both primary HSV-1 and HSV-2 infections and for diagnosis of HSV-1 encephalitis.     

HSV-1 gB and VZV gp-II crossreactive antibodies in human sera

Summary The specificity and prevalence of human IgG antibodies crossreactive between HSV-1 (ANG) and VZV (Ellen) was examined in immunoblots. Using antibody fractions purified on HSV- and VZV-coated affinity chromatography columns and by preadsorption of sera with HSV and/or VZV lysates a cross-reactivity between HSV-1 gB and VZV gp-II was demonstrated. Crossreaction of human IgG antibodies among other structural and nonstructural viral proteins, however, was not detected. The frequency of human IgG antibodies crossreactive between HSV-1 gB and VZV gp-II was highest in HSV-seropositive patients experiencing an acute primary VZV infection (4 out of 5 sera tested). In contrast, no crossreactive antibodies were found in sera of HSV-seronegative patients with acute primary VZV infection (0/6) or in sera from individuals with acute recurrent HSV or VZV infection (0/12). Analysis of sera from individuals with previous HSV and/or VZV infection showed the presence of antibodies crossreactive between HSV-1 gB and VZV gp-II in 3 out of 30 sera tested.     

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.     

Measurement of antirotavirus IgM/IgA/IgG responses in the serum samples of Indian children following rotavirus diarrhoea and their mothers

Rotavirus specific, serum IgM/IgA/IgG levels among hospitalized children and their respective mothers were determined. Children were grouped as having rotavirus diarrhoea (RVD) and non-rotavirus diarrhoea (NRVD) on the basis of fecal excretion measured by ELISA and RT-PCR. Although IgM seropositivity was observed among children of both the groups, it was significantly higher in the acute as well as convalescent phase serum samples (P < 0.05 for both) of RVD group. Five out of ten acute sera from the NRVD group were positive for IgM and seven showed IgA/IgG seroconversion indicating rotavirus infection among these children in the past. It was noted that, three out of 24 mothers' sera from RVD group, showed presence of IgM in the serum collected during convalescence of their children. The observation suggests, subclinical rotavirus infection among mothers probably contacted from their children. This is supported by the seroconversion for IgA/IgG among these three mothers. Such a phenomenon was not noticed among the mothers from NRVD group. In general, IgA positivity did not vary significantly among the children from both the groups. IgA seropositivity was significantly higher (P < 0.001) from children of RVD group as compared to healthy group of children following rotavirus infection. From RVD group, all the child patients and 12 mothers out of 24 (50%) showed IgA/IgG seroconversion. None of the mothers from NRVD group showed seroconversion. Serum samples of healthy children and adults, showed IgM positivity at equal level (10%), but a significant difference (P < 0.01) was observed in IgA positivity. In conclusion, subclinical transmission of rotavirus infection from children to their mothers may occur. Seroconversion alone cannot be considered as a marker of rotavirus diarrhoea in children. Moreover, about 40-50% of subjects lacked rotavirus specific IgA at protective levels, making them susceptible to rotavirus infection.     

Evaluation of mixed infection cases with both herpes simplex virus types 1 and 2

Herpes simplex virus type 1 (HSV-1) is isolated principally from the upper half of the body innervated by the trigeminal ganglia whereas herpes simplex virus type 2 (HSV-2) is generally isolated from the lower half of the body innervated by the sacral ganglia. However, recent reports suggest that HSV-1 and HSV-2 can each infect both the upper and lower half of the body causing a variety of symptoms and there is a possibility that HSV-1 and HSV-2 infections can occur simultaneously with both causing symptoms. HSV type in clinical isolates from 87 patients with genital herpes and 57 with ocular herpes was determined by the polymerase chain reaction (PCR), and six cases of mixed infection with both HSV-1 and HSV-2 were identified. Of the six cases, three were patients with genital herpes and three were ocular herpes patients. Analysis of the copy number of the HSV-1 and HSV-2 genome by a quantitative real time PCR demonstrated that HSV-1 was dominant at a ratio of approximately 100:1 in the ocular infections. In contrast, the HSV-2 genome was present at a 4-40 times higher frequency in isolates from genital herpes patients. There was no obvious difference between the clinical course of mixed infection and those of single HSV-1 or HSV-2 infections. This study indicated that the frequency of mixed infection with both HSV-1 and HSV-2 is comparatively higher than those of previous reports. The genome ratio of HSV-1 and HSV-2 reflects the preference of each HSV type for the target organ.     

Immunoglobulins M and G to varicella-zoster virus measured by solid-phase radioimmunoassay: antibody responses to varicella and herpes zoster infections.

Both immunoglobulin M (IgM) and IgG antibodies to varicella-zoster virus (VZV) were detectable in a solid-phase radioimmunoassay with 125I-labeled goat antisera to human immunoglobulins. Primary infection with VZV was associated with early production of IgM and IgG antibodies and rapid development of lymphocyte transformation to VZV antigen. Among eight subjects with varicella tested 1 to 4 days after onset, seven patients had IgG and six patients had IgM antibodies; all patients had both IgG and IgM antibodies within 7 days. An IgM response was documented by radioimmunoassay in 18 of 26 patients with herpes zoster. VZV antibodies could be assayed by radioimmunoassay in unfractionated serum with commercial goat antisera to human immunoglobulins and commercial VZV antigen. VZV-specific IgG binding was present in all sera from 42 subjects with a VZB antibody titer of greater than or equal to 1:8 as determined by indirect immunofluorescence and cellular immunity to VZV as determined by lymphocyte transformation and who had had varicella at least 20 years before testing. The geometric mean titer was 1:6,309, and titers were greater than or equal to 1:16,384 in 20 subjects. Antibody was present as determined by radioimmunoassay in 14 samples negative by complement fixation and in five samples negative by complement fixation and immune adherence hemagglutination. No specific binding was observed in 21 sera from subjects who were not immune to VZV as determined by indirect immunofluorescence or lymphocyte transformation despite the presence of herpes simplex or cytomegalovirus antibody indicated by complement fixation in 15 sera. High titers of VZV IgM antibody were detected in unfractionated sera despite the presence of high titers of VZV IgG antibody. The VZV radioimmunoassay provided a sensitive and practical method for measuring VZV IgG and IgM antibodies.