Part 1 of webinar: Multiplexed Serodiagnostic Test with Advanced Machine Learning Classifier for Single-Tier Diagnosis of Lyme Disease
Lyme disease is a high-incidence infectious disease with a global prevalence of 14.5% of the world's population. Two-tier serology is currently the primary diagnostic test for Lyme disease; however, this approach has limitations and does not aid in diagnosis at the earliest, most treatable stage. Here, we present an innovative multiplexed serodiagnostic test aiming to improve the diagnostic accuracy of Lyme disease testing. The assay measures both IgM and IgG antibodies against multiple Borrelia proteins and peptides utilizing Luminex xMAP® Technology, and includes a unique calibrator for quantification. Luminex’s output is interpreted with an advanced machine learning classifier to provide an unambiguous test result. The test is performed using Luminex analyzers already in place in many clinical laboratories. This test could potentially improve early diagnosis of Lyme disease, enabling earlier treatment to reduce the risk of Lyme-associated chronic illness attributed to undiagnosed cases. This single test achieves high sensitivity without losing specificity in people with both early- and late-stage Lyme disease.
Learning Objectives:
1. Discuss the challenges of diagnosing Lyme disease and the limitations of the current two-tier serological tests.
2. Explain how the combination of IgM and IgG antibody detection with a machine-learning classifier significantly improves test accuracy.
3. Compare test performance of the multiplexed immunoassay with two tier serology, and consider how improved early detection of Lyme disease would improve patient short- and long-term outcomes.
Part 2 of webinar: A Novel Blood Test for Axial Spondyloarthritis: Anti-14-3-3 eta Multiplex Test Validation
Background:
New Day Diagnostics, LLC validated the Anti-14-3-3η Multiplex Laboratory Developed Test (LDT) for precision, analytical sensitivity, specificity, accuracy, reportable range, and robustness. The assay qualitatively detects anti-14-3-3η autoantibodies against five peptide targets in human serum using xMAP® multi-analyte immunoassay technology. Elevated anti-14-3-3η levels are associated with axial spondyloarthritis (axSpA), a type of arthritis that causes inflammation in the spine and large joints that results in pain and stiffness.
Methods:
Precision followed a 20-day, 2-run-per-day, 2-replicate-per-run design (80 data points per sample). Analytical sensitivity and hook-effect studies used CLSI EP17-A2 guidelines. Clinical performance was assessed with 269 retrospective serum samples (158 axSpA, 111 healthy) and assessed by cross-validation. Accuracy, linearity, robustness, interference, and stability were evaluated under protocol-specific acceptance criteria.
Results:
Repeatability ranged 4.5–11.4 %CV and within-laboratory precision 8.3–14 %CV. Limit of Blank (LoB) & Limit of Detection (LoD) were determined for all targets. No significant hook effect or clinically relevant cross-reactivity was observed except for minor signal reduction for target 7 ≥ 160 mg/dL. Cross-validation yielded 60.2 % positive percent agreement (PPA) and 75.7 % negative percent agreement (NPA), exceeding acceptance thresholds (≥ 49.5 % PPA, ≥ 66.3 % NPA). Accuracy reached 96.0 % agreement. All targets exhibited linearity. Robustness and stability studies passed; serum remained stable ≥ 5 weeks at –80 °C, 48 h at 2–8 °C, and 48 h at room temperature.
Conclusions:
The Anti-14-3-3η Multiplex LDT met or exceeded all analytical and clinical validation criteria. With 60.2 % sensitivity, 75.7 % specificity, and 96 % accuracy, the assay demonstrates reliable performance suitable for clinical application in evaluating patients for axial spondyloarthritis.