Jespersen, Deborah J.

Shell vials (SV) and conventional tubes (CT) were seeded with rhabdomyosarcoma (RD) and MRC-5 cells and inoculated with clinical specimens, and the systems were evaluated for the rapid diagnosis of herpes simplex virus (HSV) infections by detection of cytopathic effects (CPE) (for CT, for 7 days) and by using fluoresceinated monoclonal antibodies (for SV, 16 h postinoculation). Of 245 genital specimens (16 from males and 229 from females) 56 (23%) seeded with MRC-5 cells (14 type 1 and 42 type 2) and 55 (22%) seeded with RD cells were detected in CT; however, CPE were recognized in only 26 (46%) of the total HSV-positive cultures 1 day postinoculation. Forty-eight (86% sensitivity, MRC-5) and 46 (84% sensitivity, RD) HSV strains were detected immunologically in SV 16 h postinoculation. Early CPE in CT or fluorescent foci in SV were easier to detect in MRC-5 than in RD cell cultures. MRC-5 and RD cells were equally sensitive to infection with HSV. CT cell cultures were more sensitive than SV but less rapid for the detection of HSV infection (P less than 0.01). We recommend using SV for the rapid diagnosis of HSV infections, but in addition, CT must be inoculated with MRC-5 or RD to ensure maximum detection of this virus.

ABSTRACT Detection of the Histoplasma capsulatum urinary antigen (UAg) is among the most sensitive and rapid means to diagnose histoplasmosis. Previously, we evaluated analyte-specific reagents (ASR) manufactured by IMMY (Norman, OK) for detection of Histoplasma galactomannan (GM) in urine using an enzyme immunoassay (EIA), and we showed low positive agreement (64.5%) with the MiraVista (MVista) Histoplasma antigen (Ag) quantitative EIA (MiraVista Diagnostics, Indianapolis, IN). Here we reevaluated the IMMY GM ASR following modification of our original assay protocol and introduction of an indeterminate range. A total of 150 prospectively collected urine samples were tested with both the IMMY and MVista EIAs, and clinical histories were recorded for all study subjects. The IMMY GM ASR showed positive and negative agreements of 82.3% (14/17 samples) and 100% (121/121 samples), respectively (with exclusion of 12 indeterminate results), and overall agreement of 90% (135/150 samples) with respect to the MVista EIA. Of the three patients with negative IMMY GM ASR results and positive MVista EIA results, testing was performed for initial diagnostic purposes for one patient (<0.4 ng/ml by the MVista EIA) and UAg levels were being monitored for the remaining two patients (both <0.7 ng/ml by the MVista EIA). The MVista EIA results were positive for 6/12 samples that tested indeterminate by the IMMY GM ASR. We also show that the IMMY GM ASR can be used to serially monitor Histoplasma UAg levels. In conclusion, we demonstrate that, with modification, the IMMY GM ASR is a reliable rapid assay for detection of Histoplasma UAg.

ABSTRACT Detection of Histoplasma capsulatum urinary antigen (UAg) is important for the initial diagnosis of infection and for monitoring of patient responses to antifungal therapy. This study evaluated an analyte-specific reagent (ASR) enzyme immunoassay (EIA) for the detection of H. capsulatum UAg from Immuno Mycologics, Inc. (IMMY) (Norman, OK) in comparison with routine testing with the MiraVista (MVista) H. capsulatum quantitative EIA (MiraVista Diagnostics, Indianapolis, IN). Using prospectively collected urine specimens ( n = 1,003), we observed an overall percent agreement between the two assays of 97.6% (979/1,003 samples). Compared with the MVista EIA, the sensitivity and specificity of the IMMY ASR EIA were 64.5% (40/62 samples) and 99.8% (939/941 samples), respectively, using a cutoff value of 0.5 ng/ml. Based on available clinical histories for 23/24 discordant samples, 5 IMMY assay-negative/MVista assay-positive samples were considered falsely positive. Furthermore, 10/23 discordant samples were positive by the MVista EIA but were below the limit of quantitation (<0.4 ng/ml). The clinical significance of these low positive results in the MVista EIA is unclear. In addition to the prospective study, we tested 11 urine specimens collected from patients with culture-confirmed Histoplasma infections, and 100% (11/11 samples) were positive by the IMMY ASR EIA. In conclusion, the IMMY ASR EIA may offer an alternative approach for the detection of Histoplasma UAg. Additional prospective studies are needed to better characterize the performance of the IMMY ASR EIA in conjunction with clinical and laboratory findings.

ABSTRACT We compared the performance of four assays for the detection of cryptococcal antigen in serum samples ( n = 634) and cerebrospinal fluid (CSF) samples ( n = 51). Compared to latex agglutination, the sensitivity and specificity of the Premier enzyme immunoassay (EIA), Alpha CrAg EIA, and CrAg lateral flow assay (LFA) were 55.6 and 100%, 100 and 99.7%, and 100 and 99.8%, respectively, from serum samples. There was 100% agreement among the four tests for CSF samples, with 18 samples testing positive by each of the assays.

ABSTRACT We describe the first direct comparison of the reverse and traditional syphilis screening algorithms in a population with a low prevalence of syphilis. Among 1,000 patients tested, the results for 6 patients were falsely reactive by reverse screening, compared to none by traditional testing. However, reverse screening identified 2 patients with possible latent syphilis that were not detected by rapid plasma reagin (RPR).

ABSTRACT The diagnosis of syphilis is challenging and often relies on serologic tests to detect treponemal or nontreponemal antibodies. Recently, the Centers for Disease Control and Prevention and the Association of Public Health Laboratories proposed an update to the syphilis serology testing algorithm, in which serum samples are first tested using a treponema-specific test and positive samples are analyzed with a nontreponemal assay. The goal of this study was to compare the performance of seven treponemal assays (BioPlex 2200 syphilis IgG [Bio-Rad, Hercules, CA], fluorescent treponemal antibody [FTA] assay [Zeus Scientific, Raritan, NJ], Treponema pallidum particle agglutination [TP-PA; Fujirebio Diagnostics, Malvern, PA], Trep-Sure enzyme immunoassay [EIA; Phoenix Biotech, Oakville, Ontario, Canada], Trep-Chek EIA [Phoenix Biotech], Trep-ID EIA [Phoenix Biotech], and Treponema ViraBlot IgG [Viramed Biotech AG, Planegg, Germany]) using serum samples ( n = 303) submitted to our reference laboratory. In addition to testing with these 7 assays, all samples were tested by a rapid plasma reagin (RPR) assay and a treponemal IgM Western blot assay (Viramed ViraBlot). Compared to the FTA assay as the gold standard, the evaluated treponemal tests demonstrated comparable levels of performance, with percent agreement ranging from 95.4% (95% confidence interval, 92.3 to 97.3) for the Trep-Sure EIA to 98.4% (96.1 to 99.4) for the Trep-ID EIA. Compared to a “consensus of the test panel” (defined as at least 4 of 7 treponemal tests being in agreement), the percent agreement ranged from 95.7% (92.7 to 97.5) for Trep-Sure to 99.3% (97.5 to 99.9) for Trep-ID. These data may assist clinical laboratories that are considering implementing a treponemal test for screening or confirmatory purposes.

ABSTRACTThe goal of this study was to evaluate the BioPlex 2200Toxoplasma, rubella, and cytomegalovirus (CMV) (ToRC) IgG and IgM multiplex immunoassays (Bio-Rad Laboratories, Hercules, CA) and compare the results to those of conventional testing by enzyme immunoassay (EIA) and enzyme-linked fluorescent assay (ELFA). Serum specimens (n= 600) submitted for routine ToRC IgG and IgM testing by EIA (SeraQuest, Doral, FL; Diamedix, Miami, FL) or ELFA (Vidas; bioMérieux, Durham, NC) were also tested by the BioPlex ToRC multiplex immunoassays. Samples showing discordant results were retested by both methods, with further discrepancies being arbitrated by a third assay. Following repeat testing, the BioPlexToxoplasma, rubella, and CMV IgG assays demonstrated agreements of 98.7 (592/600 specimens), 93.3 (560/600 specimens), and 98.3% (590/600 specimens), respectively, while the ToRC IgM assays yielded agreements of 91.2 (547/600 specimens), 87.3 (524/600 specimens), and 95.2% (571/600 specimens), respectively. The BioPlex ToRC IgG assays provided results comparable to EIA/ELFA results, with kappa coefficients showing near-perfect agreement for theToxoplasma(κ = 0.94) and CMV (κ = 0.97) IgG assays and substantial agreement for the rubella IgG assay (κ = 0.66). The BioPlex ToRC IgM assays showed lower specificity with only slight agreement forToxoplasmaIgM (κ = 0.07), poor agreement for rubella IgM (κ = −0.03), and moderate agreement for CMV IgM (κ = 0.55). Both the BioPlex IgG and IgM assays reduced turnaround time (1.7 h versus 5.5 h by EIA/ELFA for 100 specimens) and eliminated the necessity to manually pipette or aliquot specimens prior to testing.

ABSTRACT The laboratory diagnosis of syphilis is based primarily upon serologic findings. Historically, serologic testing for syphilis has relied on assays such as rapid plasma reagin, fluorescent treponemal antibody absorption, Treponema pallidum particle agglutination (TP-PA), and more recently, enzyme immunoassay (EIA). In this study, we evaluated the performance of a novel multiplex flow immunoassay (BioPlex 2200 Syphilis; Bio-Rad Laboratories, Hercules, CA) for the detection of antitreponemal IgG- and IgM-class antibodies. Serum specimens ( n = 1,008) submitted for routine treponema-specific antibody testing by syphilis IgM and IgG EIA (Trep-Chek; Phoenix-Biotech, Mississauga, Ontario, Canada) were also analyzed by the BioPlex Syphilis multiplex assay. Specimens showing discordant results were repeat tested, with further discrepancies being arbitrated by TP-PA. Compared directly to the results of EIA, the BioPlex IgG assay demonstrated 98.7% (77/78) sensitivity and 99.4% (916/930) specificity. Compared to the Trep-Chek IgM EIA, the BioPlex IgM assay showed 80% (4/5) sensitivity and 97.9% (652/666) specificity. These results indicate that the BioPlex Syphilis multiplex assay shows similar serological agreement with EIA while allowing for a fully automated random-access platform that provides faster (1.7 h for 100 samples versus 4.5 h by EIA) and higher-throughput (800 samples per 9 h versus 200 samples by EIA) analysis of the syphilis serologic response.

ABSTRACT 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.

ABSTRACTConventional methods for the detection of Epstein-Barr virus (EBV)-specific antibodies include the immunofluorescence assay (IFA) and enzyme immunoassay (EIA). While sensitive and specific, these methods are labor-intensive and require separate assays for each analyte. This study evaluated the performance of a multiplex bead assay (BioPlex 2200; Bio-Rad Laboratories, Hercules, CA) for the simultaneous detection of immunoglobulin G (IgG) and IgM class antibodies to the EBV viral capsid antigen (VCA) and IgG class antibodies to Epstein-Barr virus nuclear antigen-1 (EBNA-1). Serum specimens (n= 1,315) submitted for routine EBV-specific antibody testing by EIA (Grifols-Quest, Inc., Miami, FL) were also tested by the multiplex bead assay using the BioPlex 2200 automated analyzer. Specimens showing discordant results were tested by IFA. Following IFA resolution, the BioPlex VCA IgM, VCA IgG, and EBNA-1 IgG assays demonstrated 97.9%, 91.4%, and 96.9% agreement, respectively, with the results obtained by EIA. Furthermore, the BioPlex assays showed an overall agreement of 94.1% with the EIA when the specimens were categorized by disease state (susceptible, acute, or past infection) based on the EBV-specific antibody profiles. These findings indicate that the BioPlex EBV assays demonstrate a performance comparable to that of the conventional EIA, while allowing for a more rapid (2.3 h for 100 samples versus 4.5 h by the EIA) and higher-throughput (∼400 samples per 9 h versus 200 samples by the EIA) analysis of the EBV-specific antibody response.