Citations

Abstract Background Detecting Histoplasma (HP) and Blastomyces (BM) antigen (Ag) in urine (U) and serum (S) is an essential diagnostic tool for these pathogens. Labs have added bronchoalveolar lavage (BAL) and cerebrospinal fluid (CSF) as additional HP and BM Ag testing sources. We evaluated the sensitivity of Ag detection in U and S in patients with detected HP or BM Ag in BAL or CSF by enzyme immunoassay (EIA). Methods 14 patients with HP or BM Ag-positive results made up the CSF cohort. The Ag concentrations were below the limit of quantification (< LoQ, N=3), above the limit of quantification ( > LoQ, N=1), and 0.3 to 9.5 ng/mL (x̄ = 2.3 ng/mL, N=10). The BM Ag-positive in BAL group included 24 patients, with concentrations of < LoQ (N=4), >LoQ (N=5), and 0.3 to 11.3 ng/mL (x̄ = 3.5 ng/mL, N=15). HP Ag-positive in BAL comprised 36 patients, with 3 < LoQ, 10 >LoQ, and 23 ranging from 0.8 to 16.9 ng/mL (x̄ = 4.7 ng/mL). 16 patients in the BAL cohorts were Ag-positive for HP and BM, demonstrating the inability to discriminate between these dimorphic fungi through Ag testing. All patients were checked for routine clinical HP and BM EIA Ag test results on U and S. HP BAL patients were also investigated for cross-reacting galactomannan (GM; Aspergillus EIA in S or BAL) and (1→3)-β-d-glucan (BDG; Fungitell EIA in S) antigen test orders. All additional test results over 14 days before or after CSF or BAL Ag testing were excluded. Results 100% (14/14), 96% (22/23), and 83% (30/36) of HP/BM CSF, BM BAL, and HP BAL Ag-positive patients were also positive in U and/or S, when both additional sources were ordered, respectively. Of the 6 HP BAL patients without positive U or S results, 3 had GM and BDG testing performed. All 3 had high signal GM and/or BDG results in S and/or BAL. In conjunction with low HP Ag levels in BAL (1.0 to 1.53), these findings suggest cross-reactivity in the HP BAL Ag test. Classifying these 3 patients as HP BAL false-positive shows a positive agreement of 91% (30/33) between HP BAL and HP U/S. Conclusion HP and BM CSF Ag testing showed no increase in sensitivity when U and S were tested. Minimal increases in sensitivity were observed in HP and BM BAL versus U and S. However, false-positive results in BAL due to cross-reactivity with GM and BDG cannot be ruled out, suggesting limited utility of the invasive CSF and BAL sources for HP and BM EIA Ag testing. Disclosures All Authors: No reported disclosures

Abstract Background Diagnosis of VZV CNS vasculitis (CNS-VZV) is aided by detecting intrathecally (i.t.) synthesized VZV-specific antibodies (Abs) through testing paired serum (S) and CSF specimens and determining an antibody index (AI). Currently, laboratories in the United States do not provide AI testing for VZV. The currently used methods (e.g., EIAs, IFAs) lack clinical utility as they cannot discriminate if VZV Abs in the CSF are i.t. produced due to CNS infection or whether they are present due to passive diffusion across the blood-CSF-barrier. To optimize diagnostic options for CNS-VZV, we evaluated a quantitative VZV IgG ELISA (Euroimmun AG, Lübeck, Germany) to calculate AI values using Reiber’s formula on paired S/CSF specimens to assess the diagnostic accuracy of this method. Methods The clinical sensitivity cohort consisted of four patients diagnosed with CNS-VZV (meningitis or vasculitis) as determined by symptoms, imaging, response to antiviral therapy, and/or supporting test results (e.g., lymphocytic pleocytosis, elevated protein in CSF). The patient group for clinical specificity included 11 patients with suspected CNS-VZV who ultimately had an unclear (n=4) or non-CNS-VZV (n=7) diagnosis. The reference range cohort included paired clinical S/CSF samples from 25 patients without infectious diseases testing. Paired S/CSF samples were processed on the same batch using a fully automated ELISA analyzer capable of onboard, ad hoc runtime dilution preparation (AP22 ELITE, DAS ITALY). The AI values were calculated using the Reiber formula, and sensitivity (Sn), specificity (Sp), and positive (PPV) and negative (NPV) predictive values were calculated. The AI was calculated using the following equations: If Total IgG CSF (mg/dL):Total IgG serum (mg/dL) < Quotient limit Total IgG, then AI = (VZV IgG Ab CSF [units × dilution]:VZV IgG serum [units × dilution])/(Total IgG CSF [mg/dL]:Total IgG serum [mg/dL]). If Total IgG CSF (mg/dL):Total IgG serum (mg/dL) > Quotient limit Total, then AI = (VZV IgG Ab CSF [units × dilution]:VZV IgG serum [units × dilution])/Quotient limit Total IgG. Quotient limit Total IgG = 0.93 × [(Qalbumin2) + (6 × 10-6)]1/2 - 1.7 ×10-3. Qalbumin = Albumin CSF (mg/dL):Albumin serum (mg/dL). Results The VZV IgG ELISA with Reiber AI calculation method showed Sn, Sp, PPV, and NPV values of 100% (4/4), 91% (10/11), 80% (4/5), and ?0 (10/10), respectively. One of the Sp patients had an equivocal AI result and was classified as negative, whereas the Sp patient with a positive AI result had an unclear diagnosis. VZV AI results for 96% (24/25) of the reference range patients were negative, with one equivocal. The mean AI of the 25 patients was 1.05, and the lower and upper 3 standard deviation limits were 0.6 and 1.5, respectively. These values match the AI qualitative interpretive criteria recommended by Reiber (0.6 to <1.3 = negative, 1.3 to 1.5 = equivocal, >1.5 AI = positive). Conclusion We show that the VZV IgG ELISA, alongside the Reiber formula for AI calculation, is effective for identifying patients with i.t. synthesized VZV IgG Abs. This method can assist clinicians in evaluating patients with suspected CNS-VZV infections who are negative by alternative methods.

ABSTRACT 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 ~10 10±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 .

ABSTRACT 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.

Abstract Background Diagnosis of neurosyphilis (NS) is challenging due to nonspecific clinical symptoms and the lack of a sensitive and specific laboratory test. The non-treponemal Venereal Disease Research Laboratory (VDRL-CSF) assay is a commonly used test in diagnosing NS. An important limitation of the VDRL-CSF is limited specificity, in part due to the inability to differentiate between intrathecally produced antibodies vs passive antibody diffusion into the CSF due to blood-brain barrier (BBB) disruption. Here, we compared the intrathecal synthesis antibody index (AI) of anti-Treponema pallidum IgG antibodies to results from the VDRL-CSF assay in routinely submitted clinical samples for evaluation of suspected NS. Methods Paired serum and CSF residual clinical samples from 98 patients with previous VDRL-CSF results were collected. For each pair, the serum and CSF were drawn within 24 h. No additional clinical information and limited supporting clinical test results were available. Thirty-one positive and 67 negative paired samples were tested by an anti-T. pallidum IgG ELISA (Euroimmun, Lübeck, Germany). The AI was calculated using Reiber’s method, with intrathecal total IgG synthesis corrected using the Reiber recommended albumin quotient calculation. Results were compared for positive, negative and overall agreement between methods. Results The anti-T. pallidum AI showed a positive, negative and overall agreement of 71.0% (22/31), 100% (67/67) and 90.8% (89/98), respectively, compared to VDRL-CSF. Of note, all nine discordant samples showed abnormally elevated albumin levels in CSF (range: 27.4 to 215 mg/dl; median = 41.7 mg/dl; normal value =&lt;27 mg/dl), indicating a compromised BBB which likely resulted in false-positive VDRL-CSF results. The range and median value for CSF albumin in the 22 concordant samples was 12.4 to 168 mg/dl and 37.2 mg/dl, respectively. In total, 83.9% (26/31) of VDRL-CSF positive samples showed elevated CSF albumin levels which suggests a high percentage of BBB impairment in this patient population. This demonstrates the advantage in specificity that is offered by AI testing compared to use of the VDRL-CSF assay, given the ability of AI to differentiate intrathecally produced vs passively diffused syphilis-specific antibodies. No potential increase in sensitivity was observed in AI testing as 100% negative agreement was obtained. Conclusion Our data suggests that establishment of an AI using a treponemal assay to detect intrathecal antibody synthesis provides improved specificity compared to the current commonly performed VDRL-CSF antibody test as an aid in the diagnosis of NS. Further evaluation of sensitivity is warranted using samples from patients with clinically diagnosed NS.

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.

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.

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.

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.

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.