Phage K exposure generates phage-neutralizing activity in a rabbit model-humoral receptiveness may impact efficacy of phage therapy.

Phage therapy has not been established in the clinical routine, in part due to uncertainties concerning efficacy and immunogenicity. Here, three rabbits were immunized against staphylococcal phage K to assess viral potency in the presence of immunized serum. Three rabbits received weekly intramuscular injections of ~1010±1 pfu/mL phage K. Phage K-specific IgG formation was measured by an enzyme-linked immunosorbent assay (ELISA); phage inactivation was assessed by calculating K-rates. Using transmission electron microscopy (TEM) and immunogold labeling, antibody binding to phage K was visualized. This was numerically assessed by objective imaging analysis comparing the relative distances of each gold particle to the nearest phage head and tail structure. Immunization led to a strong IgG response, plateauing 7 days after the last phage injection. There was no significant correlation between K-rate and antibody titer over time. TEM showed IgG binding to the head structure of phage K. Image analysis showed a significant reduction in relative distances between antibodies and phage head structures when comparing samples from day 0 and day 28 (P < 0.0001). These results suggest that while individual serum analysis for antibodies against therapeutic phage bears consideration prior to and with prolonged therapy, during phage application, the formation of specific antibodies against phage may only partially explain decreased phage potency in the presence of immunized serum. Instead, other factors may contribute to an individual's "humoral receptiveness" to phage therapy. Future investigations should be directed toward the identification of the humoral factors that have the most significant predictive value on phage potency in vivo.

Detection of Histoplasma capsulatum and Blastomyces dermatitidis antigens in serum using a single quantitative enzyme immunoassay.

Histoplasma and Blastomyces antigen detection assays are commonly used diagnostic tools. However, a high level of cross-reactivity between these antigens prevents definitive pathogen identification by these assays alone. Retrospective analysis of 3,529 patients with Histoplasma and Blastomyces antigen testing performed on the same serum sample yielded an overall percent agreement of 99.3% (3,506 of 3,529; kappa: 0.859) between the two assays, suggesting that use of a single assay to detect both antigens may be an alternative diagnostic approach. We assessed performance of the Gotham BioTech Blastomyces antigen (GBA) enzyme immunoassay (EIA) (Portland, Maine) for detection of Blastomyces and Histoplasma antigens in serum. Comparison to the MiraVista Diagnostics Blastomyces (MVB) EIA showed 100% positive (24 of 24), negative (57 of 57), and overall (81 of 81) percent agreement. Additionally, 171 sera were used to compare the GBA EIA to the MiraVista Diagnostics Histoplasma (MVH) EIA, which showed 91.3% (63 of 69), 98% (100 of 102), and 95.3% (163 of 171) positive, negative, and overall percent agreement, respectively. Among eight patients with discordant GBA/MVH EIA results, seven had additional fungal testing performed, and results suggested that the MVH and GBA results were inaccurate for two and five samples, respectively. Overall, this study suggests that the GBA EIA has a high level of agreement with both of the commonly used, individual Blastomyces and Histoplasma antigen EIAs. By taking advantage of the high level of cross-reactivity between Blastomyces and Histoplasma antigen EIAs, utilization of a single antigen detection assay for these fungi provides an opportunity to optimize test utilization and decrease patient cost while maintaining a high level of diagnostic accuracy.

Limited Correlation between SARS-CoV-2 Serologic Assays for Identification of High-Titer COVID-19 Convalescent Plasma Using FDA Thresholds.

In August 2020, the Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for COVID-19 convalescent plasma (CCP) specified 12 authorized serologic assays and associated assay-specific cutoff values for the selection of high-titer CCP for use in hospitalized patients. The criteria used for establishing these cutoff values remains unclear. Here, we compare the overall agreement and concordance of five serologic assays included in the August 2020 FDA EUA at both the manufacturer-recommended qualitative cutoff thresholds and at the FDA-indicated thresholds for high-titer CCP, using serum samples collected as part of the CCP Expanded Access Program (EAP). The qualitative positive percent agreement (PPA) across assays ranged from 92.3% to 98.8%. However, the high-titer categorization across assays varied significantly, with the PPA ranging from 26.5% to 82.7%. The Roche anti-NC ECLIA provided the lowest agreement compared to all other assays. Efforts to optimize high-titer cutoffs could reduce, although not eliminate, the discordance across assays. The consequences of using nonstandardized assays are apparent in our study, and the high-titer cutoffs chosen for each assay are not directly comparable to each other. The generalized findings in our study will be relevant to any future use of convalescent plasma for either COVID-19 or future pandemics of newly emerged pathogens. IMPORTANCE COVID-19 convalescent plasma (CCP) was one of the first therapeutic options available for the treatment of SARS-CoV-2 infections and continues to be used selectively for immunosuppressed patients. Given the emergence of novel SARS-CoV-2 variants which are resistant to treatment with available monoclonal antibody (MAb) therapy, CCP remains an important therapeutic consideration. The FDA has released several emergency use authorizations (EUA) that have specified which serological assays can be used for qualification of CCP, as well as assay-specific cutoffs that must be used to identify high-titer CCP. In this study, a cohort of donor CCP was assessed across multiple serological assays which received FDA EUA for qualification of CCP. This study indicates a high degree of discordance across the assays used to qualify CCP for clinical use, which may have precluded the optimal use of CCP, including during clinical trials. This study highlights the need for assay standardization early in the development of serological assays for emerging pathogens.

Detection of neutralizing antibodies against multiple SARS-CoV-2 strains in dried blood spots using cell-free PCR.

An easily implementable serological assay to accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies is urgently needed to better track herd immunity, vaccine efficacy and vaccination rates. Herein, we report the Split-Oligonucleotide Neighboring Inhibition Assay (SONIA) which uses real-time qPCR to measure the ability of neutralizing antibodies to block binding between DNA-barcoded viral spike protein subunit 1 and the human angiotensin-converting enzyme 2 receptor protein. The SONIA neutralizing antibody assay using finger-prick dried blood spots displays 91-97% sensitivity and 100% specificity in comparison to the live-virus neutralization assays using matched serum specimens for multiple SARS-CoV-2 variants-of-concern. The multiplex version of this neutralizing antibody assay, using easily collectable finger-prick dried blood spots, can be a valuable tool to help reveal the impact of age, pre-existing health conditions, waning immunity, different vaccination schemes and the emergence of new variants-of-concern.

Multicenter Clinical Evaluation of Modified Two-Tiered Testing Algorithms for Lyme Disease Using Zeus Scientific Commercial Assays.

Modified two-tiered testing (MTTT) algorithms for Lyme disease (LD), which involve the sequential use of orthogonal enzyme immunoassays (EIAs) without immunoblotting, are acceptable alternatives to standard two-tiered testing (STTT; EIA followed by immunoblots) provided the EIAs have been FDA-cleared for this intended use. We evaluated four Zeus Scientific LD EIAs used in two distinct MTTT algorithms for FDA review. MTTT 1 used a VlsE1/pepC10 polyvalent EIA followed by a whole-cell sonicate (WCS) polyvalent EIA. MTTT 2 used the same first-tier EIA followed by separate IgM and IgG WCS EIAs. In a retrospective phase, we compared each MTTT algorithm to STTT using archived samples from LD patients or control subjects. In a prospective phase, we used the same algorithms to analyze consecutive excess samples submitted for routine LD serology to three clinical laboratories. For the retrospective phase, MTTTs 1 and 2 were more sensitive (56% and 74%) than STTT (41%; P ≤ 0.03) among 61 patients with acute erythema migrans (EM). In LD patients with neuroborreliosis, carditis, or arthritis (n = 75), sensitivity was comparable between algorithms (96 to 100%; P = 1.0). Among 190 control subjects without past LD, all algorithms were highly and comparably specific (≥99%, P = 0.48). For the prospective phase, (n = 2,932), positive percent-agreement (PPA), negative percent-agreement (NPA), and overall agreement of MTTT 1 with STTT were 93%, 97.7% and 97.4% (kappa 0.80). MTTT 2 yielded higher PPA (98%) but lower NPA (96.1%) and overall agreement (96.2%, kappa 0.74; all P < 0.05). Compared with STTT, both MTTT algorithms provided increased sensitivity in EM patients, comparable sensitivity in later disease and non-inferior specificity.

Antigen Specific Humoral and Cellular Immunity Following SARS-CoV-2 Vaccination in ANCA-Associated Vasculitis Patients Receiving B-Cell Depleting Therapy.

Humoral vaccine responses are known to be suboptimal in patients receiving B-cell targeted therapy, and little is known about vaccine induced T-cell immunity in these patients. In this study, we characterized humoral and cellular antigen-specific anti-SARS-CoV2 responses following COVID-19 vaccination in patients with ANCA-associated vasculitis (AAV) receiving anti-CD20 therapy, who were either B-cell depleted, or B-cell recovered at the time of vaccination and in normal control subjects. SARS-CoV-2 anti-spike (S) and anti-nucleocapsid (NC) antibodies were measured using electrochemiluminescence immunoassays, while SARS-CoV-2 specific T-cell responses to S glycoprotein subunits 1 (S1) and 2 (S2) and receptor binding domain peptide pools were measured using interferon-gamma enzyme-linked immunosorbent spot (ELISPOT) assays. In total, 26 recently vaccinated subjects were studied. Despite the lack of a measurable humoral immune response, B-cell depleted patients mounted a similar vaccine induced antigen-specific T-cell response compared to B-cell recovered patients and normal controls. Our data indicate that to assure a humoral response in patients receiving anti-CD20 therapy, SARS-CoV-2 vaccination should ideally be delayed until B-cell recovery (CD-20 positive B-cells > 10/µl). Nevertheless, SARS-CoV-2 vaccination elicits robust, potentially protective cellular immune responses in these subjects. Further research to characterize the durability and protective effect of vaccine-induced anti-SARS-CoV-2 specific T-cell immunity are needed.

Detection of Blastomyces dermatitidis Antigen in Urine Using a Commercially Available Quantitative Enzyme Immunoassay.

Laboratory diagnosis of blastomycosis relies on a combination of methods, including antigen detection. We assessed the performance of analyte-specific reagents from Gotham Biotech (Portland, ME) for quantitative detection of Blastomyces dermatitidis galactomannan (GM) in urine using an enzyme immunoassay (EIA) compared to the Blastomyces quantitative EIA from MiraVista Diagnostics (Indianapolis, IN). Residual urine from 232 unique patients previously tested by the MiraVista assay was evaluated using the Gotham EIA, which showed 97.4% (74/76), 100% (156/156), and 99.1% (230/232) positive, negative, and overall agreement, respectively. Correlation between the quantitative B. dermatitidis antigen levels by the Gotham and MiraVista EIAs was low (R2 = 0.20). Medical records were available for 36 of the 232 patients, among whom four had confirmed blastomycosis and both the Gotham and MiraVista EIAs were positive. Nine of these patients had histoplasmosis, and the Gotham and MiraVista EIAs yielded negative results in 44.4% (4/9) and 22.2% (2/9) of cases, respectively. Both assays were negative in the remaining 23 patients. After laboratory implementation of the Gotham EIA, chart reviews were performed on the first 50 unique patients (51 samples) tested by the assay in our hospital. Among these, 3/50 (6%) samples were positive by the Gotham EIA, including two samples from a patient with culture-confirmed blastomycosis and one from a patient with histoplasmosis (also positive by the MiraVista Blastomyces EIA). All remaining patients were negative by the Gotham EIA and had alternative diagnoses. Our findings show comparable performance between the Gotham and MiraVista quantitative EIAs for detection of B. dermatitidis GM in urine.

SARS-CoV-2 Serologic Assays Dependent on Dual-Antigen Binding Demonstrate Diverging Kinetics Relative to Other Antibody Detection Methods.

Longitudinal studies assessing durability of the anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) humoral immune response have generated conflicting results. This has been proposed to be due to differences in patient populations, the lack of standardized methodologies, and the use of assays that measure distinct aspects of the humoral response. SARS-CoV-2 antibodies were serially measured in sera from a cohort of 44 well-characterized convalescent plasma donors over 120 days post-COVID-19 symptom onset, utilizing eight assays, which varied according to antigen source, the detected antibody isotype, and the activity measured (i.e., binding, blocking, or neutralizing). While the majority of assays demonstrated a gradual decline in antibody titers over the course of 120 days, the two electrochemiluminescence immunoassay Roche assays (Roche Diagnostics Elecsys anti-SARS-CoV-2 [qualitative, nucleocapsid based] and Roche Diagnostics Elecsys anti-SARS-CoV-2 S [semiquantitative, spike based]), which utilize dual-antigen binding for antibody detection, demonstrated stable and/or increasing antibody titers over the study period. This study is among the first to assess longitudinal, rather than cross-sectional, SARS-CoV-2 antibody profiles among convalescent COVID-19 patients, primarily using commercially available serologic assays with Food and Drug Administration emergency use authorization. We show that SARS-CoV-2 antibody detection is dependent on the serologic method used, which has implications for future assay utilization and clinical value.

Detection of SARS-CoV-2 IgG antibodies in dried blood spots.

The COVID-19 pandemic led to development of numerous serologic tests. To obviate the need for phlebotomy services, we validated dried blood spots (DBS) for anti-SARS-CoV-2 serologic testing. Immunoglobulins were extracted from 3 mm DBS punches and the extracts were analyzed using the Euroimmun anti-SARS-CoV-2 IgG ELISA. Various pre-analytical factors were studied. Results were favorable for DBS stored for at least 67 days at or below 37°C. Comparison between paired serum and DBS specimens tested by the Euroimmun ELISA showed 96.8% and 81.3% positive and negative agreement, respectively, indicating that confirmatory testing of positive Euroimmun results on DBS extracts is necessary to achieve clinical accuracy. Our findings suggest that any SARS-CoV-2 antibody assay that requires pre-dilution of serum is amenable to DBS as an alternate specimen type that is relatively low cost, self-collectable, stable, can be shipped by standard mail and could be used to establish the seroprevalence of large populations.

Validation of a multiplex flow immunoassay for detection of IgG antibodies against SARS-CoV-2 in dried blood spots.

BACKGROUND: Dried blood spots (DBS) are an established specimen type for clinical testing given their low cost, ease of collection and storage, and convenient shipping capabilities through the postal system. These attributes are complementary to the expansion of SARS-CoV-2 serologic testing, which may be used to inform community seroprevalence rates. METHODS: The Luminex xMAP SARS-CoV-2 Multi-Antigen assay utilizes magnetic beads labeled with three viral antigens (nucleocapsid [NC], receptor binding domain [RBD], spike S1 subunit) to detect anti-viral IgG-class antibodies, and has Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for use in serum and plasma. This assay was modified for use with DBS and validated against paired sera tested by one of two reference assays: the Roche Diagnostics Elecsys anti-SARS-CoV-2 ECLIA or the Euroimmun anti-SARS-CoV-2 IgG ELISA. RESULTS: 159 paired DBS and serum specimens analyzed using the modified Luminex xMAP assay on DBS and the reference methods on serum showed an overall concordance of 96.9% (154/159). Use of multivariate pattern recognition software (CLIR) for post-analytical interpretation of the Luminex xMAP DBS assay results, instead of manufacturer provided interpretive thresholds, increased overall qualitative result concordance to 99.4% (158/159) between the modified Luminex xMAP DBS and reference results. CONCLUSIONS: Use of DBS for detection of antibodies against SARS-CoV-2 provides comparable results to those obtained using serum. DBS concordance was improved with multivariate pattern recognition software (CLIR). We demonstrate that DBS are a reliable specimen type for SARS-CoV-2 antibody detection using the modified Luminex xMAP assay.

Prevalence of SARS-CoV-2 Antibodies in a Multistate Academic Medical Center.

OBJECTIVE: To estimate the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in health care personnel. METHODS: The Mayo Clinic Serology Screening Program was created to provide a voluntary, two-stage testing program for SARS-CoV-2 antibodies to health care personnel. The first stage used a dried blood spot screening test initiated on June 15, 2020. Those participants identified as reactive were advised to have confirmatory testing via a venipuncture. Venipuncture results through August 8, 2020, were considered. Consent and authorization for testing was required to participate in the screening program. This report, which was conducted under an institutional review board-approved protocol, only includes employees who have further authorized their records for use in research. RESULTS: A total of 81,113 health care personnel were eligible for the program, and of these 29,606 participated in the screening program. A total of 4284 (14.5%) of the dried blood spot test results were "reactive" and warranted confirmatory testing. Confirmatory testing was completed on 4094 (95.6%) of the screen reactive with an overall seroprevalence rate of 0.60% (95% CI, 0.52% to 0.69%). Significant variation in seroprevalence was observed by region of the country and age group. CONCLUSION: The seroprevalence for SARS-CoV-2 antibodies through August 8, 2020, was found to be lower than previously reported in other health care organizations. There was an observation that seroprevalence may be associated with community disease burden.

Serum Epitope Repertoire Analysis Enables Early Detection of Lyme Disease with Improved Sensitivity in an Expandable Multiplex Format.

Widely employed diagnostic antibody serology for Lyme disease, known as standard two-tier testing (STTT), exhibits insufficient sensitivity in early Lyme disease, yielding many thousands of false-negative test results each year. Given this problem, we applied serum antibody repertoire analysis (SERA), or next-generation sequencing (NGS)-based serology, to discover IgG and IgM antibody epitope motifs capable of detecting Lyme disease-specific antibodies with high sensitivity and specificity. Iterative motif discovery and bioinformatic analysis of epitope repertoires from subjects with Lyme disease (n = 264) and controls (n = 391) yielded a set of 28 epitope motifs representing 20 distinct IgG antibody epitopes and a set of 38 epitope motifs representing 21 distinct IgM epitopes, which performed equivalently in a large validation cohort of STTT-positive samples. In a second validation set from subjects with clinically defined early Lyme disease (n = 119) and controls (n = 257), the SERA Lyme IgG and IgM assay exhibited significantly improved sensitivity relative to STTT (77% versus 62%; Z-test; P = 0.013) and improved specificity (99% versus 97%). Early Lyme disease subjects exhibited significantly fewer reactive epitopes (Mann-Whitney U test; P < 0.0001) relative to subjects with Lyme arthritis. Thus, SERA Lyme IgG and M panels provided increased accuracy in early Lyme disease in a readily expandable multiplex assay format.

Performance Characteristics of Four High-Throughput Immunoassays for Detection of IgG Antibodies against SARS-CoV-2.

The role of serologic testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in both 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, 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 coronavirus disease 2019 (COVID-19), healthy donor sera from 2018, and a cross-reactivity serum panel collected in early 2020. The sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical IgG assays in convalescent-phase serum samples collected more than 14 days post-symptom onset or post-initial positive reverse transcriptase PCR (RT-PCR) result were 92.9% (78/84), 88.1% (74/84), 97.6% (82/84), and 98.8% (83/84), respectively. Among unique convalescent patients, sensitivities of the Abbott, Epitope, Euroimmun, and Ortho-Clinical anti-SARS-CoV-2 IgG assays were 97.3% (36/37), 73% (27/37), 94.6% (35/37), and 97.3% (36/37), respectively. Overall assay specificity/positive predictive values based on a 5% prevalence rate were 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-phase 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.

Performance Characteristics of a Multiplex Flow Immunoassay for Detection of IgG-Class Antibodies to Measles, Mumps, Rubella, and Varicella-Zoster Viruses in Presumptively Immune Health Care Workers.

Immunity to measles, mumps, rubella, and varicella-zoster viruses (VZV; MMRV) is a common condition of employment for health care workers (HCWs) to ensure compliance with national standards and state laws. When documentation of complete vaccination or laboratory-confirmed infection is not available, Advisory Committee on Immunization Practices (ACIP) criteria are used to guide vaccination or anti-MMRV IgG testing. We assessed the performance of the BioPlex 2200 MMRV IgG multiplex flow immunoassay (MFI; Bio-Rad Laboratories, Hercules, CA) and matched immunofluorescence assays (IFAs; MBL Bion, Des Plaines, IL) in 220 HCWs categorized by ACIP criteria for presumptive immunity to MMRV. Among HCWs presumptively immune to measles, mumps, rubella, and VZV, the Bio-Rad MFI was positive in 77.3, 85.4, 84.3, and 91.1% of HCWs, respectively. Comparatively, the Bion IFA was positive in 92.9, 91.1, and 93.5% of HCWs presumptively immune to measles, mumps, and VZV (a rubella IFA was unavailable). Among HCWs fully vaccinated against measles, mumps, and VZV, Bio-Rad MFI/Bion IFA positivity rates were 77.4%/93%, 84.8%/90.7%, and 54.5%/90.9%, respectively. The Bio-Rad MFI was positive in 83.7% of HCWs fully vaccinated against rubella. For HCWs whose last vaccination event occurred within 15 years of enrollment, 83.3, 93.3, and 74.2% were positive by the Bio-Rad measles, mumps, and rubella IgG MFIs, respectively. We show significantly decreased Bio-Rad MFI sensitivity for detection of anti-measles and anti-mumps IgG-class antibodies in presumptively immune or fully vaccinated HCWs. Although negative results typically prompt revaccination, failure to recognize presumptive immunity in individuals unable to receive live, attenuated vaccines may have employment implications.

Evaluation of a Rapid Immunochromatographic Assay and Two Enzyme-Linked Immunosorbent Assays for Detection of IgM-Class Antibodies to Zika Virus.

There are currently five serologic assays available for detection of anti-Zika virus (ZIKV) IgM-class antibodies with U.S. Food and Drug Administration emergency use authorization. Among these are the Chembio DPP Zika IgM system (DPP Zika ICA; Chembio, Medford, NY), a rapid immunochromatographic assay (ICA), and the InBios ZIKV Detect 2.0 IgM antibody capture enzyme-linked immunosorbent assay (ZIKV 2.0 MAC-ELISA; InBios international, Inc., Seattle, WA), which has replaced the original InBios ZIKV Detect MAC-ELISA. We evaluated performance of these three serologic assays using 72 specimens characterized by plaque reduction neutralization testing (PRNT) for the presence or absence of neutralizing antibodies (NAbs) to ZIKV, dengue virus (DENV), and West Nile virus (WNV). The InBios ZIKV 2.0 MAC-ELISA was "presumptive Zika positive" in all 15 PRNT-confirmed ZIKV samples, while the Chembio DPP Zika ICA was nonreactive in three (20%) and the InBios ZIKV MAC-ELISA was negative in four (27%). The Chembio DPP Zika ICA and InBios ZIKV 2.0 MAC-ELISA showed >95% specificity in 22 ZIKV/DENV-seronegative specimens and in 13 samples positive for NAbs to non-ZIKV flaviviruses. Comparatively, the InBios ZIKV MAC-ELISA was "presumptive" or "possible Zika positive" in 8 of 12 WNV or DENV PRNT-positive samples and in 12 of 22 PRNT-seronegative sera. Our findings suggest that replacement of the InBios ZIKV MAC-ELISA with the InBios ZIKV 2.0 MAC-ELISA will lead to fewer samples requiring PRNT, minimizing unnecessary anxiety among patients ultimately determined to be seronegative for ZIKV and DENV by PRNT and alleviating some of the testing burden on laboratories performing PRNT.

Evaluation of a Novel Microarray Immunoblot Assay for Detection of IgM- and IgG-Class Antibodies to Borrelia burgdorferi.

Diagnostic testing for Lyme disease (LD) remains dependent on detection of antibodies to LD Borrelia using serologic assays, in adherence to the standard two-tiered testing (STTT) algorithm. We present the first analytic evaluation of the automated Borrelia B31 ViraChip IgM and IgG microarray immunoblot (MIB) assays (Viramed Biotech AG, Planegg, Germany) in comparison to two different, semiautomated blot assays for LD, including the Borrelia B31 ViraStripe IgM and IgG line immunoassays (LIAs) (Viramed) and the MarDx Borrelia burgdorferi IgM and IgG Western blot (WB) assays (Trinity Biotech, Carlsbad, CA), using prospectively collected sera (n = 411) and archived, clinically characterized samples (n = 91). We show comparable overall agreement (>84%) of the ViraChip MIB assays against the two aforementioned LD blot methods. The ViraChip MIB assays were also compared to a consensus standard, whereby samples were classified as positive or negative for IgM or IgG to B. burgdorferi if the analyte-matched ViraStripe LIA or MarDx WB assay were positive or negative, respectively. The ViraChip IgM and IgG MIB assays showed >93% positive, negative, and overall agreement versus these consensus criteria. The ViraChip MIB assays were associated with a time savings of 28 min to process one full batch of samples compared to the time required for the ViraStripe LIAs. The ViraChip MIB assays can be programmed and performed on an open-system, automated enzyme-linked immunosorbent assay (ELISA) processor, negating the need for assay-specific equipment and enabling laboratories to consolidate LD testing onto a single platform. We conclude that the ViraChip IgM and IgG MIB assays may be added to the repertoire of supplemental, second-tier blot testing systems for diagnosis of LD.

An Unusual Cluster of Neuroinvasive Lyme Disease Cases Presenting With Bannwarth Syndrome in the Midwest United States.

Bannwarth syndrome (BWS), an infrequent manifestation of neuroinvasive Lyme disease (LD) characterized by radiculopathy, neuropathy, and lymphocytic pleocytosis, is more commonly documented in Europe than North America. Here, we describe a cluster of 5 neuroinvasive LD cases with BWS in the upper Midwest United States between July and August 2017.

Low Cryptococcus Antigen Titers as Determined by Lateral Flow Assay Should Be Interpreted Cautiously in Patients without Prior Diagnosis of Cryptococcal Infection.

Detection of Cryptococcus antigen (CrAg) is invaluable for establishing cryptococcal disease. Multiple different methods for CrAg detection are available, including a lateral flow assay (LFA). Despite excellent performance of the CrAg LFA, we have observed multiple cases of low-titer (≤1:5) positive CrAg LFA results in patients for whom cryptococcosis was ultimately excluded. To investigate the accuracy of low-titer positive CrAg LFA results, we performed chart reviews for all patients with positive CrAg LFA results between June 2014 and December 2016. During this period, serum and/or cerebrospinal fluid (CSF) samples from 3,969 patients were tested with the CrAg LFA, and 55 patients (1.5%) tested positive. Thirty-eight of those patients lacked a history of cryptococcal disease and were the focus of this study. Fungal culture or histopathology confirmed Cryptococcus infection for 20 patients (52.6%), and CrAg LFA titers in serum and CSF samples ranged from 1:5 to ≥1:2,560. For the 18 patients (47.4%) without culture or histopathological confirmation, the CrAg LFA results were considered true-positive results for 5 patients (titer range, 1:10 to ≥1:2,560), due to clinical improvement with targeted therapy and decreasing CrAg LFA titers. The remaining 13 patients had CrAg LFA titers of 1:2 (n = 11) or 1:5 (n = 2) and were ultimately diagnosed with an alternative condition (n = 11) or began therapy for possible cryptococcosis without improvement (n = 2), leading to an overall CrAg LFA false-positive rate of 34%. We recommend careful clinical correlation prior to establishing a diagnosis of cryptococcal infection for patients with first-time positive CrAg LFA titers of 1:2.

Serologic Testing for Zika Virus: Comparison of Three Zika Virus IgM-Screening Enzyme-Linked Immunosorbent Assays and Initial Laboratory Experiences.

Serologic evaluation for Zika virus (ZIKV) infection currently includes an initial screen using an anti-ZIKV IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) followed by supplemental testing of specimens with nonnegative results by a plaque reduction neutralization test (PRNT). We compared the performance characteristics of three ELISAs for the detection of IgM class antibodies to ZIKV, including the Centers for Disease Control and Prevention (CDC) Zika MAC-ELISA, the InBios ZIKV Detect MAC-ELISA, and the Euroimmun anti-Zika Virus IgM ELISA. Additionally, we present our initial experiences with ZIKV serologic testing from a national reference laboratory perspective. Using both retrospectively and prospectively collected specimens from patients with possible ZIKV infection, we show that the CDC and InBios MAC-ELISAs perform comparably to each other, with positive agreement, negative agreement, and interrater kappa values ranging from 87.5% to 93.1%, 95.7% to 98.5%, and 0.52 to 0.83, respectively. In contrast, comparison of the Euroimmun ZIKV ELISA to either the CDC or InBios MAC-ELISAs resulted in positive agreement, negative agreement, and interrater kappa values ranging from 17.9% to 42.9%, 91.7% to 98.6%, and 0.10 to 0.39, respectively. Among the 19 prospective samples submitted for PRNT, nine were negative, eight specimens had neutralizing antibodies to a flavivirus (unable to be identified), and one sample each was confirmed for ZIKV or dengue virus infection. This study highlights the ongoing challenges associated with serologic diagnosis of ZIKV infection. Although the availability of a commercial serologic test for ZIKV has greatly expanded the national capacity for such testing, the need to further characterize and improve these assays, particularly with regard to specificity, remains.