The role of serologic testing for SARS-CoV-2, both in the clinical and public health settings, will continue to evolve as we gain increasing insight into our immune response to the virus. Here, we evaluated four high throughput serologic tests for detection of anti-SARS-CoV-2 IgG antibodies, including assays from Abbott Laboratories (Abbott Park, IL), Epitope Diagnostics Inc. (San Diego, CA), Euroimmun (Lubeck, Germany), and Ortho-Clinical Diagnostics (Rochester, NY), using a panel of serially collected serum samples (N=224) from 56 patients with confirmed COVID-19, healthy donor sera from 2018 and a cross-reactivity serum panel collected in early 2020. Sensitivity of the Abbott, Epitope, Euroimmun and Ortho-Clinical IgG assays in convalescent serum samples collected more than 14 days post symptom onset or initial positive RT-PCR result was 92.9% (78/84), 88.1% (74/84), 97.6% (82/84) and 98.8% (83/84), respectively. Among unique convalescent patients, sensitivity of the Abbott, Epitope, Euroimmun and Ortho-Clinical anti-SARS-CoV-2 IgG assays was 97.3% (36/37), 73% (27/37), 94.6% (35/37) and 97.3% (36/37), respectively. Overall assay specificity and positive predictive values based on a 5% prevalence rate are 99.6%/92.8%, 99.6%/90.6%, 98.0%/71.2% and 99.6%/92.5%, respectively, for the Abbott, Epitope, Euroimmun and Ortho-Clinical IgG assays. In conclusion, we show high sensitivity in convalescent sera and high specificity for the Abbott, Euroimmun and Ortho-Clinical anti-SARS-CoV-2 IgG assays. With the unprecedented influx of commercially available serologic tests for detection of antibodies against SARS-CoV-2, it remains imperative that laboratories thoroughly evaluate such assays for accuracy prior to implementation.

The goal of this study was to evaluate the BioPlex 2200 Toxoplasma, rubella, and cytomegalovirus (CMV) Congenital infections caused by Toxoplasma gondii, rubella, and cytomegalovirus (CMV) are a significant cause of neonatal mortality and childhood morbidity worldwide (6,18,21). Due to their nonspecific clinical manifestations and the importance of early recognition of in utero infection, serologic screening for these pathogens has been considered a routine practice in many parts of the world (11). Conventional methods for the detection of antibodies to Toxoplasma, rubella, and CMV (ToRC) include immunofluorescence (IFA), enzyme immunoassay (EIA), and enzyme-linked fluorescent assay (ELFA). These techniques have been used for years in both diagnostic and screening protocols for ToRC infection and have demonstrated reliable performance (5,10,14,22). However, these methods have certain limitations, including low throughput, significant hands-on time, and in the case of IFA, a subjective interpretation of results.(Recently, multiplex flow immunoassay (MFI) technology emerged as a novel approach to assess the serologic response to various infectious diseases (3, 4, 13). This technology is similar to traditional EIA but allows for the simultaneous detection and identification of multiple analytes in a single reaction. MFI technology uses a liquid suspension array of up to 100 unique microspheres (5-to 6-m beads), each conjugated with a different capture molecule (e.g., antibody, antigen, 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. In 2009, Bio-Rad Laboratories (Hercules, CA) received FDA clearance for a ToRC IgG immunoassay based on MFI technology. In addition, Bio-Rad has developed a prototype ToRC IgM assay for use in cases of suspected acute infection. These assays are fully automated on the BioPlex 2200 automated analyzer (Bio-Rad Laboratories), allowing for a high-throughput analysis of the ToRC IgG and IgM class antibody response.Due to increasing test volumes (ϳ20% in the past 5 years) and the limitations of conventional methods (e.g., low throughput, increased hands-on time, and the requirement to aliquot samples prior to testing), we undertook a study to evaluate the BioPlex ToRC IgG and IgM immunoassays using clinical serum samples. The goal of this study was to compare the results of the BioPlex to routine testing by EIA/ELFA, using a third assay to arbitrate discordant results. MATERIALS AND METHODSStudy design. Prospective serum specimens (n ϭ 600) submitted to our reference laboratory 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 IgM and IgG immunoassays using the BioPlex 2200 automated analyzer (Bio-Rad). Other specimen types, including cord blood samples, were not included in this evaluation. Specimens showing discordant res...

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. Localized largely along the Ohio River and Mississippi River valleys, Histoplasma capsulatum continues to be a significant cause of morbidity and death, particularly among individuals with weakened cellular immunity and those over the age of 65 years (1, 2). Despite the high rates of exposure (60 to 90%) among residents of these regions of endemicity, otherwise healthy individuals are able to control infections with minimal disease manifestations (3, 4). However, immunocompromised patients exposed to H. capsulatum typically present with a nonspecific febrile illness, which can rapidly progress to pneumonia, respiratory insufficiency, disseminated disease, and death (2, 5). Histoplasmosis can be treated effectively with proper antifungal management, but timely diagnosis is essential.Due to the nonspecific clinical presentation of histoplasmosis, as well as lengthy, costly, and potentially toxic therapeutic regimens, laboratory testing to confirm the diagnosis is critical. Many methods are available for this purpose, including culture, nucleic acid amplification testing (NAAT), histopathological examination, and antibody and antigen (Ag) detection. The individual performance characteristics of these methods vary, with their overall clinical utility being largely dependent on disease presentation (localized versus disseminated), level of patient immunosuppression, and symptom duration prior to testing. Briefly, wh...

Conventional 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 (ϳ400 samples per 9 h versus 200 samples by the EIA) analysis of the EBV-specific antibody response. Epstein-Barr virus (EBV) is the primary agent of infectious mononucleosis (IM), a common syndrome characterized by fever, pharyngitis, and lymphadenopathy. Most individuals become infected during childhood, and it is estimated that nearly 95% of the adult population worldwide is seropositive for the virus (20). While the majority of infections result in either asymptomatic or mild disease, serious complications, including B-and T-cell lymphomas, nasopharyngeal carcinoma, and central nervous system involvement, may occur, especially in immunocompromised hosts (14).The diagnosis of IM is made, in most cases, on the basis of characteristic clinical manifestations or the detection of heterophile antibodies (24). However, a determination of the EBVspecific antibody response may be required for young children (especially those Ͻ4 years old) (26) and for adults suspected of having heterophile-negative IM. Testing for immunoglobulin M (IgM) and IgG class antibodies to the viral capsid antigen (VCA) and for IgG class antibodies to Epstein-Barr virus nuclear antigen-1 (EBNA-1) allows for a discrimination between recent and remote infection (8,18,21,22). Levels of antibodies (IgM and/or IgG) to VCA are typically elevated during the acute phase of IM (19,22,26), with anti-VCA IgM levels showing a steady decline 4 to 6 weeks after symptom onset (16). In contrast, anti-VCA IgG persists indefinitely, and its detection along with that of anti-EBNA-1 IgG suggests past exposure to the virus (25)...

Detection of