Herpes simplex virus type 2 antibody detection performance in Kisumu, Kenya, using the Herpeselect ELISA, Kalon ELISA, Western blot and inhibition testing.

BACKGROUND: In certain parts of Africa, type-specific herpes simplex virus type 2 (HSV-2) ELISAs may have limited specificity. To date, no study has been conducted to validate HerpeSelect and Kalon type-specific HSV-2 ELISAs using both the Western blot and recombinant gG ELISA inhibition testing as reference standards. METHODS: A total of 120 men who were HIV seronegative (aged 18-24 years) provided blood samples. HSV-2 IgG serum antibodies were detected using four different methods: HerpeSelect HSV-2 ELISA (n = 120), Kalon HSV-2 ELISA (n = 120), University of Washington Western blot (n = 101) and a recombinant inhibition test (n = 93). RESULTS: HSV-2 seroprevalence differed significantly by HSV-2 detection method, ranging from 24.8% with the Western blot to 69.8% with the HerpeSelect ELISA. Using the Western blot as the reference standard, the HerpesSelect had the highest sensitivity for HSV-2 antibody detection (100%) yet lowest specificity (40%). Similar results were obtained using the inhibition test as the reference standard. The sensitivity and specificity of the Kalon test versus the Western blot were 92% and 79%, respectively, and 80% and 82% versus the inhibition test. Using the inhibition test as the reference standard, the sensitivity of the Western blot appeared low (49%). CONCLUSIONS: In men in western Kenya who were HIV seronegative, the HerpeSelect and Kalon type-specific ELISAs had high sensitivities yet limited specificities using the Western blot as reference standard. Overall, the Kalon ELISA performed better than the HerpeSelect ELISA in these young men from Kisumu. Further understanding is needed for the interpretation of HSV-2 inhibition or ELISA test positive/ Western blot seronegative results. Before HSV-2 seropositivity may be reliably reported in selected areas of Africa, performance studies of HSV-2 serological assays in individual geographical areas are recommended.

Evaluation of an enzyme immunoassay system for measuring herpes simplex virus (HSV) type 1-specific and HSV type 2-specific IgG antibodies.

MRL Diagnostics has developed a dual enzyme immunoassay (EIA) system that employs the recombinant Herpes Simplex Virus (HSV) type-specific glycoproteins G1 (HSV1) and G2 (HSV2) to detect HSV type-specific IgG antibodies. This system was evaluated using 155 consecutive sera previously tested in a conventional dual EIA system (Zeus) that employs multiple HSV1 and HSV2 proteins to detect type-common as well as type-specific antibodies. Sera were also analyzed by Western blot to determine the true HSV type-specific IgG reactivity pattern. Of 110 sera giving concordant reactivity patterns in the MRL and Zeus EIA systems, 108 (98%) also displayed concordant Western blot patterns; two sera gave false positive HSV2 reactivity in both EIA systems. Of 45 sera giving discordant MRL and Zeus EIA reactivity patterns, 41 (91%) displayed a Western blot reactivity pattern that matched the MRL reactivity pattern. Both the HSV1 IgG component and the HSV2 IgG component of the MRL EIA system were 100% sensitive and > 95% specific. In contrast, the Zeus HSV1 IgG EIA was 98% sensitive and 79% specific, and the Zeus HSV2 IgG EIA was 85% sensitive and 79% specific. An analysis of the distribution of index values in the MRL EIA system showed that low-positive values (1.0-3.0) were rare, but, when detected, often represented false positive results; only 11 MRL low-positive results were observed, but all 6 MRL false positive results were found within this low-positive subgroup. These findings show that the MRL dual EIA system effectively detects HSV type-specific IgG antibodies.

Whole cell lysate enzyme immunoassays vs. recombinant glycoprotein G2-based immunoassays for HSV-2 seroprevalence studies.

Seroepidemiology studies of herpes simplex virus type 2 (HSV-2) infections have been difficult to carry out because antibodies to HSV type 1 (HSV-1) show an extensive cross-reactivity with HSV-2 antigens. Many kits available currently are not entirely type specific for serodiagnosis of HSV-2 infections and therefore do not allow reliable discrimination of past exposure to these closely related alphaherpes viruses. Attempts to develop type-specific antigens have focused on the envelope glycoproteins, particularly glycoprotein G (gG). A cross-sectional study was carried out to examine the seroprevalence of antibodies to HSV-2 among healthy university students, using different methods: a whole cell lysate enzyme-linked immunosorbent assay (ELISA), two different ELISAs, and a newly developed immunoblot assay, the last three based on recombinant gG2. HSV-2 prevalence was 24 times higher with the whole cell lysate ELISA (31%; 95% confidence interval [CI]: 27-35%) than the ELISAs and the immunoblot assay based on recombinant gG2 (1.3%; 95% CI: 0.1-2.5%), thus showing the inaccuracy of commercial tests based on whole-antigen preparations for epidemiological studies. Laboratories should be cautious and ensure that commercial tests for HSV typing are based on type-specific glycoproteins.

Evaluation of a line immunoblot assay for detection of antibodies recognizing extractable nuclear antigens.

Sera (n = 90) giving positive results in a screening test for antibodies to extractable nuclear antigens (ENAs) were tested in a line immunoblot assay that measures antibody reactivity with individual ENAs in a single test field. Results were then compared to those obtained in monospecific ENA antibody enzyme immunoassays (EIAs). Discordant results were resolved by immunodiffusion. Of 540 result pairs (90 sera tested for 6 ENAs [Sm/RNP, Sm, SSA, SSB, Scl-70, Jo-1]), 509 (94%) showed concordance. Immunodiffusion resolved 28 of 31 discordant result pairs in favor of the immunoblot result. After resolution of discordant data, the immunoblot assay exhibited 100% sensitivity for all ENA antibodies except those recognizing Scl-70, for which the sensitivity was 89%; specificity was over 96% for all 6 ENA antibodies. These findings show that a line immunoblot assay for the characterization of ENA antibodies yields results comparable to those obtained using monospecific ENA antibody EIAs. The immunoblot assay is easier and less expensive to perform due to its utilization of a single test field.

Parvovirus B19-antibody serosurvey in 500 developmentally disabled subjects.

Sera of 500 residents were screened for parvovirus B19 immunoglobulin M (IgM) and immunoglobulin G (IgG) by enzyme-linked immunosorbent assay. Positive IgM and equivocal IgG or IgM results were confirmed by immunofluorescent antibody and Western blot. IgM was detected in 13 sera (2.6%), and IgG was detected in 285 (57%). Records of IgM-positive residents contained no evidence of erythema infectiosum or polyarthropathy.

Serodiagnosis of Lyme borreliosis by Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii western blots (immunoblots).

The performance of Western blots (immunoblots) prepared with eight strains of Borrelia burgdorferi representing B. burgdorferi sensu stricto, B. garinii, and B. afzelii genospecies was tested with a panel of sera with various clinical presentations collected from eight geographic regions. European sera were generally more reactive to blots prepared with B. garinii or B. afzelii strain antigens, in particular B. garinii 20047 and B. afzelii VS461. North American sera were more reactive with B. burgdorferi sensu stricto strains. Our observation of significant differences in the levels of reactivity of some sera on Western blots of certain strains is potentially important for the development and implementation of generic interpretive criteria. Preferential reactivity of sera from patients with nerve and/or palsy symptoms to B. garinii strains and with cutaneous disease to B. afzelii strains was observed. On the basis of our results, we have concluded that strain 20047 is the best strain to use for the development of a generic Lyme borreliosis Western blot for Europe.

Childhood idiopathic thrombocytopenic purpura: association with human parvovirus B19 infection.

PURPOSE: Infection with human parvovirus B19 is the most common cause of transient aplastic crisis in patients with chronic hemolytic anemia. Multiple reports of children with simultaneous B19 infection and thrombocytopenia as well as the known association between experimental B19 infection and thrombocytopenia prompted us to hypothesize that B19 may be associated with childhood idiopathic, or immune, thrombocytopenic purpura (ITP). Because there is a paucity of evidence regarding a viral etiology for ITP, we performed a comprehensive study to explore its possible relationship to B19 infection. PATIENTS AND METHODS: Thirty-five previously healthy children with ITP were studied prospectively. Bone marrow and peripheral blood were analyzed for B19 DNA using the polymerase chain reaction (PCR). Serum was analyzed for anti-B19 immunoglobulin (Ig) M and IgG antibodies using a B19 VP1 antigen-based enzyme-linked immunosorbent assay. Fourteen healthy children served as controls for peripheral blood PCR and serologic analyses. RESULTS: The presenting clinical and laboratory features of the study population were typical of classic ITP. Seventeen of the 35 patients (49%) had evidence of B19 DNA in the peripheral blood, bone marrow, or both. Six of 35 (17%) had anti-B19 IgM antibodies. Eight of 35 (23%) were anti-B19 IgG seropositive. The control group had no positive PCR or anti-B19 IgM specimens. CONCLUSIONS: Our results suggest that infection with human parvovirus B19 may be associated with childhood ITP. More investigation is warranted regarding the role of PCR methodology and serologic detection methods in defining B19 pathobiology as it relates to ITP.

Recurrent erythema migrans despite extended antibiotic treatment with minocycline in a patient with persisting Borrelia burgdorferi infection.

Erythema migrans recurred in a patient 6 months after a course of treatment with minocycline for Lyme disease. Polymerase chain reaction on heparinized peripheral blood at that time demonstrated the presence of Borrelia burgdorferi-specific DNA. The patient was seronegative by Lyme enzyme-linked immunosorbent assay but showed suspicious bands on Western blot. Findings of a Warthin-Starry stain of a skin biopsy specimen of the eruption revealed a Borrelia-compatible structure. Reinfection was not believed to have occurred. Further treatment with minocycline led to resolution of the erythema migrans.

Immunoglobulins in hemophilia: correlation between IgG subclasses, IgE and HIV antibody.

Twenty-two patients with hemophilia who had received factor VIII concentrate were evaluated. Only 12 of 22 were seropositive for HIV. Elevation of IgG, IgG1, IgM and IgE was not related to HIV seropositivity. Means of IgG1, IgG2, and IgG3 were significantly higher in patients with elevated IgG. Means of IgG2 and IgG3 were significantly lower in patients with elevated (greater than or equal to 251) serum IgM. Seven of the 22 patients demonstrated elevated (greater than or equal to 100) serum IgE; mean serum IgA was significantly lower, though in normal range, in these seven patients. Alterations in serum immunoglobulins in patients with Hemophilia are frequently seen, however, like other immune-dysfunction in these patients, these abnormalities can not be attributed to their HIV status.

Biotreatment of s-triazine-containing wastewater in a fluidized bed reactor.

Mixed cultures of microorganisms immobilized on sand were used to degrade s-triazine-containing industrial wastewater in a fluidized bed reactor. Immobilized cell concentrations of up to 18 g/L volatile suspended solids could be achieved with the s-triazines as sole nitrogen source for growth and carbon sources added at a C--N ratio of about 12. Maximal removal efficiencies of 80% of the s-triazines could be maintained only if (a) the bio-film thickness was limited to avoid oxygen deficiency and (b) the carbon source and complete wastewater (/=20-25 h.

Biological treatment specific for an industrial wastewater containing s-triazines.

Mixed cultures of bacteria grew in medium containing real s-triazine wastes as nitrogen source. About 80% of the s-triazine waste could be degraded as determined by HPLC and by measurements of dissolved nitrogen. The culture required an added carbon source in order to degrade s-triazines. A temperature optimum near 40 degrees C was observed and a salt concentration above about 4% markedly retarded growth and the degradation of s-triazines. This system was examined as a biological treatment for wastes from syntheses of s-triazines.

Small-cell lymphoma and Sjögren's syndrome. Lymphoplasmacytic subvariant of small-cell lymphoma with IgA/kappa immunoglobulin surface markers.

To our knowledge, the association of the plasmacytoid variety of small-cell lymphoma with primary Sjögren's syndrome has not been reported. We describe a patient with SS who developed plasmacytoid small-cell lymphoma. As opposed to the commonly detected IgM, the lymphocyte surface immunoglobulins contained monoclonal IgA/kappa light chains. In addition to the unique immunopathologic features, the impressive response to chemotherapy and the importance of surface immunoglobulin markers in the diagnosis of malignancy in Sjögren's syndrome are discussed.

Virus-enhanced modulation of cell surface antigens: effect on immune lytic susceptibility.

Monkey kidney cells, upon progressive subculture, became refractory to complement (C)-dependent immune cytolysis by anti-cell serum. Arbovirus infection restored these cells to a state of lytic susceptibility. Similar results were also abtained with antibody-dependent cellular cytotoxicity (ADCC), which is C independent. Antibodies raised against different subcultures varied considerably in lytic efficiency, indicating changing patterns of host cell expression during continous subculture. Taken together with the fact that arbovirus infection festored the lytic efficiency of all antibody preparations to the same degree suggested some form of host cell antigen re-expression as a mechanism. The results obtained in several exploratory experiments indicated that the antigenic re-expression responsible for the restoration of lysis was probably a local or selective rather than a generalized phenomenon. Thus, the amount of host cell surface antigen, measured by the use of mouse anti-cell serum and 125I anti-mouse globulin, was identical in both uninfected lytic susceptible and refractory cells, and decreased in both functional states following infection. Further, the binding of 125I concanavalin A, used to quantify surface glycoproteins, was similar in both lytic refractory and susceptible cells, and in both cases declined folowing virus infection. This result was incompatible with gross "masking" of cell surface antigens by exuberant production of surface coat material in lytic resistant cells. Finally, brief trypsinization of lytic resistant cells yielded an 8-fold increase in immune lysis, a result further consistent with local rather than generalized surface changes. The data were discussed interms of modulation of cell surface antigens affected both by repeated subculture and arboviral infection, and as a possible in vitro correlate of altered self-reactivity.

Detection of dengue cell-surface antigens by peroxidase-labeled antibodies and immune cytolysis.

The appearance of dengue-specific plasma membrane (DSPM) antigens in infected LLC-MK(2) cell cultures was studied by (51)Cr release in immune cytolysis and at an ultrastructural level using peroxidase-labeled antibodies. DSPM antigen was first detected at 36 h with electron microscopy in approximately 30% of the cells, and this percentage did not increase with time. However, both surface staining with peroxidase-labeled antibodies and (51)Cr release indicated that the amount of DSPM antigen per cell increases with time. The appearance of (51)Cr release in immune cytolysis experiments with dengue-infected cells occurred much later than the peak of infectious virus release. This was in sharp contrast to immune cytolysis with a group A arbovirus, Eastern equine encephalitis, in which the kinetics of release of infectious virus and (51)Cr release were identical. This suggests different mechanisms of insertion of viral plasma membrane antigens in Eastern equine encephalitis and dengue-infected LLC-MK(2) cells.