Positive Percent Agreement Research Articles

Abstract B028: Analytical validation of tumor-informed whole genome sequencing analyses for detection of molecular residual disease in solid tumors

Abstract Introduction: Across all patients diagnosed with solid tumors, approximately two-thirds initially present with locoregional disease and are potentially eligible for curative-intent intervention. However, there is a significant subset of patients for which residual tumor cells remain afterwards, potentially leading to disease recurrence. It has been reported that these residual tumor cells, representative of molecular residual disease (MRD), can be detected through analyses of circulating tumor DNA (ctDNA), often earlier than can be achieved through radiographic imaging or clinical presentation. Methods: Labcorp Plasma Detect was utilized to analyze patient-matched tumor, germline (white blood cells), and cell-free or contrived DNA samples through whole genome sequencing at approximately 80x, 40x, and 30x depth, respectively. Raw sequencing data were demultiplexed and trimmed for read quality, then aligned to the hg19 human reference genome. Tumor-specific single nucleotide variants were identified from the tumor and germline datasets and used to determine the presence of cancer within cell-free or contrived DNA samples through a random forest machine learning model. ctDNA status was determined based on the level of signal compared to a reference cohort of noncancerous donor plasma samples (n=80). Results: Analytical specificity was determined through analysis of five noncancerous donor plasma samples with five replicates each, evaluated against 60 somatic mutation profiles obtained from patients with stage III colon cancer, demonstrating a specificity of 99.4% (835/840). Analytical sensitivity was assessed using three contrived cell line models from breast and melanoma tumors, analyzed across three tumor content levels with five replicates at each level and resulted in a limit of detection of 0.005% tumor content using a 100% hit-rate model. Precision, repeatability, and reproducibility were demonstrated within and across runs, operators, and instruments, resulting in a 100% detection rate at 0.05% tumor content with a coefficient of variation of 8.5%. Finally, analytical accuracy compared to an independent tumor-informed ctDNA MRD approach was determined through analysis of 39 clinical cases obtained from patients with bladder, breast, colorectal, and lung cancer, yielding a positive percent agreement of 91% (10/11) and negative percent agreement of 100% (24/24). The prognostic value of ctDNA MRD was demonstrated in stage III colon cancer and clinical validation results for HPV-negative head and neck squamous cell carcinoma will be presented separately. Conclusions: There is significant potential for tumor-informed, non- bespoke MRD approaches for ctDNA detection across a broad range of solid tumor types and curative-intent clinical settings. These data demonstrate the analytical performance of Labcorp Plasma Detect in a CAP/CLIA laboratory for pan-solid tumor research use together with investigational use in early stage colon cancer to support the interventional MEDOCC-CrEATE clinical trial. Citation Format: Kaitlin Victor, Andrew Georgiadis, Ellen Verner, Antoine Simmons, David Riley, James R White, Christopher Greco, Liam Cox, Jesse Fox, Jennifer B Jackson, Eric A Severson, Brian J Caveney, Marcia Eisenberg, Taylor J Jensen, Shakti Ramkissoon, Samuel V Angiuoli, Amy Greer, Kenneth Valkenburg, Mark Sausen. Analytical validation of tumor-informed whole genome sequencing analyses for detection of molecular residual disease in solid tumors [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B028.

Comparison of QIAstat-Dx and BioFire FilmArray Gastrointestinal Panels in a Pediatric Population

Accurate laboratory diagnosis of gastroenteritis is important to ensure that patients receive appropriate treatment and proper isolation precautions. This study evaluated the performance of the QIAGEN QIAstat-Dx gastrointestinal panel (QGP) in comparison to the bioMerieux BioFire FilmArray gastrointestinal panel (BGP) for the detection of gastrointestinal pathogens in 110 pediatric patients being evaluated for gastroenteritis at our hospital. We compared 23 different bacterial, viral, and parasite enteropathogens detected by the QGP against the BGP. The overall positive percent agreement (PPA) for all compared targets was 96.2% and the overall negative percent agreement (NPA) for all compared targets was 99.7%. Our study shows that QIAstat-Dx QGP provides comparable results to the BioFire BGP in our pediatric population. Additionally, the PCR cycle threshold (Ct) value reported by the QGP is potentially a helpful tool in estimating the load of the detected pathogen in stool samples.

Comparison of 2 Sets of Immunoassays Used in Modified 2-Tiered Testing Algorithms for the Diagnosis of Lyme Disease.

Since 2019, modified 2-tiered testing (MTTT) algorithms have been available for the diagnosis of Lyme disease. MTTTs replaced the standard algorithms that utilized enzyme immunoassays and immunoblots with sequential enzyme immunoassays that detect different antigens. We compared the performance of serological assays from ZEUS Scientific Inc. and DiaSorin Inc. that are used for the diagnosis of Lyme disease. Serological results were compared with clinical information gathered by chart review. Percent positive agreement (PPA) and percent negative agreement (PNA) for total immunoglobulin G (IgG)/immunoglogulin M (IgM) (n = 120) were 64% (95% confidence interval 54% to 73%) and 100% (87% to 100%), respectively. PPA and PNA for IgG (n = 93) were 91% (80% to 97%) and 66% (52% to 78%), respectively. PPA and PNA for IgM (n = 93) were 75% (62% to 85%) and 95% (82% to 99%), respectively. Fewer positive total IgG/IgM results confirmed positive for either IgG or IgM for ZEUS compared to DiaSorin. Overall MTTT algorithm interpretation was concordant in 58% (55/95) of samples, and concordance improved when the results were limited to IgM in patients with symptom duration <30 days. Treatment with antibiotics was most strongly associated with IgM positivity. This analysis highlights differences in the performance characteristics between commercially available diagnostic assays for Lyme disease. Our data suggest that the DiaSorin assays would result in fewer positive total IgG/IgM tests, decreasing the required number of confirmatory IgG and IgM tests. This would potentially lead to fewer patients treated with antibiotics.

Accuracy of point-of-care SARS-CoV-2 detection using buccal swabs in pediatric emergency departments.

To optimize the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected children, specimen collection and testing method are crucial considerations. Ideally, specimen collection is easy and causes minimal discomfort, and the laboratory approach is simple, accurate, and rapid. In this prospective cohort study we evaluated the accuracy of a point-of care nucleic acid device using caregiver/patient self-collected buccal swabs. Participants were recruited in 14 Canadian tertiary care pediatric emergency departments. Children <18 years of age deemed to require SARS-CoV-2 testing were eligible. Caregivers or the patient-collected buccal swabs which were tested on the ABBOTT ID NOW. The reference standard was nasopharyngeal swab specimens collected by a healthcare provider tested via laboratory reverse transcription PCR (RT-PCR). We enrolled 2,640 study participants and 14.4% (381/2,640) were SARS-CoV-2 RT-PCR-positive. Eight percent (223/2,640) and 85.0% (2,244/2,640) were concordant test-positive and concordant test-negative, respectively. Sensitivity and specificity of the investigational approach were 58.5% [95% confidence interval (CI): 53.4, 63.5] and 99.3% (95% CI: 98.9, 99.6), respectively. Cycle threshold values were lower among concordant [median 17 (15, 21)] relative to discordant [median 30 (22, 35)] swabs (P < 0.001). Sensitivity was greatest among children <4 years of age, when caregivers performed the swabs, among unvaccinated children, and those with shorter symptom duration. Across multiple pain measures, less pain was associated with buccal swab testing. Although accuracy of the buccal swab point-of-care SARS-CoV-2 test was good and negative agreement was excellent, sensitivity was only 58.5%. Concordance was greater among those with higher viral loads, and the approach involving buccal swabs was less painful.IMPORTANCETo optimize the identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected children, specimen collection and testing method are crucial considerations. Ideally, specimen collection is easy and causes minimal discomfort, and laboratory approach is simple, accurate, and rapid. We evaluated the accuracy and pain associated with buccal swab specimen collection by caregivers or children themselves who were tested using a point-of-care isothermal nucleic acid amplification SARS-CoV-2 test. This novel approach was compared to nasopharyngeal swab specimens tested using laboratory-based PCR tests. While negative agreement was excellent, positive percent agreement was less than 60%. Concordance was greater among those with higher viral loads, and thus, sensitivity is excellent when transmissibility is more likely to occur. Importantly, the approach involving buccal swabs was significantly less painful, and thus, children and their caregivers are more likely to agree to testing using such an approach.CLINICAL TRIALSRegistered at ClinicalTrials.gov (NCT05040763).

Clinical Performance of Semi-Automated Spectral-Domain Optical Coherence Tomography Angiography

Background/Objectives: To evaluate the clinical performance of two optical coherence tomography angiography (OCTA) devices, including a semi-automated device, with respect to image quality and pathology detection, with fluorescein angiography (FA) and indocyanine green angiography (ICGA) serving as the reference standards. Methods: In this prospective cross-sectional study, normal eyes and those with various retinal and choroidal pathologies were enrolled and underwent OCTA scanning using semi-automated 3D OCT-1 Maestro2 and Cirrus™ HD-OCT 5000 devices, as well as FA/ICGA imaging. OCTA scans and FA/ICGA images were independently graded for image quality and the visibility of prespecified anatomic vascular features, along with the presence or absence of pathology on the OCTA scans and the FA/ICGA images (within regions corresponding to the OCTA scan areas). Positive percent agreement (PPA), defined as the proportion of eyes in which the OCTA demonstrated pathology when the corresponding FA/ICGA showed pathology, and negative percent agreement (NPA), defined as the proportion of eyes in which the OCTA showed no pathology when the FA/ICGA also showed no pathology, were calculated. Results: In total, 38 normal eyes and 86 pathologic eyes were enrolled in the study. The majority of images for both devices were considered clinically useful. The PPA and NPA were high for both devices, indicating a good ability to identify disease when present and to rule it out when not present. Conclusions: The findings of this study suggest that the semi-automated Maestro2 and Cirrus have comparably good clinical performance, particularly with regard to accuracy when identifying vascular pathologies.

Clinical Validation and Comparison of Bacterial Vaginosis Detection

Abstract Introduction/Objective Bacterial vaginosis (BV) is considered a top reason for women’s gynecological visits, causing symptoms such as vaginal discharge, foul odor, itching, and burning associated with the alteration of the normal vaginal microbiome. It is often presented as a decrease in Lactobacillus spp. and an increase in anaerobic organisms such as Gardnerella vaginalis and Atopobium vaginae. In response to the demand for more automated and less subjective testing, molecular methods have recently been developed to aid in BV testing. The objectives of this study are to validate and compare the molecular Aptima® Bacterial Vaginosis Assay by Hologic® to the established BV Screen via a Gram stained vaginal smear graded through Nugent scoring at RUSH University Medical Center in Chicago, IL and perform a cost analysis between the testing methods. Methods/Case Report Samples of known BV value were tested on the Panther® instrument to validate the Aptima® Bacterial Vaginosis Assay for accuracy and precision. Clinical samples of vaginal swabs were used to make a Gram stained BV screen for Nugent scoring, then afterwards were placed into Hologic Multitest Aptima® probes for molecular testing on the Panther® instrument. Nugent scoring was performed blind by the molecular test. Molecular results of negative, positive, and invalid were then compared to the Nugent scores: scores of 0-3 for negative/normal vaginal flora, 7-10 for positive BV, and 4-6 for intermediate/altered scores were screened for discrepancy. Results (if a Case Study enter NA) The Aptima® BV Assay tested 52 known samples with 97% accuracy and 100% precision. A total of 80 clinical samples of vaginal swabs used for Gram stained BV screens were run on the Aptima® BV Assay; 61 provided conclusive data for comparative analysis. When compared to the original Nugent scores, the positive percent agreement (PPA) was 77.0% and negative percent agreement (NPA) was 92.9%. Conclusion The Aptima® BV Assay proved to be both accurate and precise in its validation testing. Clinically, it was able to screen for BV in vaginal samples with a good percent agreement to Gram stained BV screens. Further testing of additional clinical samples are to be done to continue analysis. Although the supplies needed to perform a Gram stain cost less than molecular testing, the reimbursement payment for molecular testing is much greater and overall reduces employee time costs for performing the test, making it the more cost effective choice for the laboratory.

Use of T2Bacteria Panel in Pediatric Hematopoietic Stem Cell Transplant Patients: A Single Center Experience

Abstract Background Bloodstream infection is a common complication following hematopoietic cell transplant (HCT), occurring in 10-40% of patients (1,2). Rapid identification of causative bacteria is imperative to provide proper empiric therapy and ensure optimal patient outcomes. The T2Bacteria Panel detects five bacterial pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli) using T2 magnetic resonance (T2MR, T2Biosystems, Lexington MA, USA) directly from blood specimens. Previous studies have reported per-patient sensitivity, specificity, and negative predictive value (NPV) of 84-90%, 85.9-90%, and 99.7%, respectively, as well as 100% positive agreement and 98.4% negative agreement when compared to blood culture (3-5). To date, there have been no studies on the use of this test in pediatric HCT recipients. Methods We reviewed all pediatric HCT patients at St. Jude Children’s Research Hospital who had a T2Bacteria Panel performed for fever and suspected infection between January 2019 and September 2022. A total of 706 T2Bacteria Panel results and concurrent blood culture results from 251 unique patients were analyzed to determine assay performance for detection of the 5 targeted pathogens. Results T2Bacteria PCR and blood culture showed concordant results in 97% of cases. Overall, our study revealed 65% positive percent agreement with blood culture and 95% negative percent agreement for bacteremia caused by any of the target pathogens. PPV, NPV, sensitivity and specificity for each organism detected by the panel, and the panel overall, are presented in Table 1. Positive predictive value ranged from 28-73%. Negative predictive value was high for all organisms (98-100%). Sensitivity was highest for Pseudomonas aeruginosa (88%), and lowest for Escherichia coli (44%). Specificity was high for all organisms (97-99%). Conclusion In pediatric HSCT patients, the T2Bacteria Panel was most useful for rapidly ruling out the presence of target pathogens, with high negative percent agreement, NPV, and specificity. Positive percent agreement was lower than noted in previous studies. Positive predictive value and sensitivity varied per pathogen.

Evaluation of the Seegene Allplex™ RV master assay for one-step simultaneous detection of eight respiratory viruses in nasopharyngeal specimens

BackgroundThe Seegene Allplex™ RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene’s multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. ObjectiveWe aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Study designDiagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the Allplex™ SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. ResultsSensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7 % to 100 % for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9 %, while the reproducibility PPA, NPA and OPA between independent operators were all 100 %. ConclusionThese results demonstrate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores the diagnostic utility of the RVM assay in detecting eight viral respiratory pathogens.

B-079 Characterization of an isoelectric focusing method for detection of IgG oligoclonal banding in paired cerebrospinal fluid and serum specimens

Abstract Background Qualitative assessment of IgG-specific oligoclonal banding (OCB) in paired serum and cerebrospinal fluid (CSF) samples is useful for evaluating inflammatory conditions of the central nervous system such as multiple sclerosis. Here we characterized the performance of an IgG-specific method using isoelectric focusing on agarose gel matrix for the detection of OCB in paired serum and CSF samples. Methods Specimens of 20 paired, simultaneously collected CSF and serum patient samples were tested on Sebia (Hydrasys 2 System) provided by the National Institutes of Health (NIH) Diagnostic Laboratories. We tested these paired samples using the Helena IgG IFE-20 kit (SPIFE® Touch) method and the results were read by two independent individuals blinded to each other’s assessments. The results were compared to the NIH results for agreement. Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were calculated. Analytical sensitivity of the Helena assay was assessed via serial dilutions of a CSF OCB positive sample (total IgG 4.5 mg/dL). Results Figure 1 shows serum and CSF pairs for the control (A), two example positive OCB patients (B &amp; D) and a negative OCB patient (C). The Helena assay was concordant with Sebia for 11/12 positive OCB patients (PPA 91.6%) and 8/8 negative subjects (NPA 100%). 20-fold was the highest dilution that remained positive. Conclusions OCB detection using paired serum and CSF samples and isoelectric focusing electrophoresis is considered the preferred method. The Helena IgG IFE-20 assay is such a method and showed high PPA and NPA concordance with the Sebia System. Thus, the Helena OCB assay can be considered an accurate and analytically sensitive method for assessing OCB in serum and CSF.

B-073 Analytical Performance of a Novel, Fully Automated Multiplexed Microarray Immunoassay Prototype for the Simultaneous Detection of Fifteen Autoantibodies Associated with Connective Tissue Diseases

Abstract Background There is currently a limited offer of automated multi-analyte tests for autoantibody testing in autoimmune connective tissue diseases (CTD). We assessed the analytical performance of a novel, single-use, multiplexed microarray immunoassay prototype (MosaiQ AiPlex CTDplus), used with the fully automated MosaiQ® system, for the simultaneous detection of fifteen autoantibodies associated with CTD, compared with selected CE-marked devices. Methods Banked human serum samples, characterized as reactive to ≥1 autoantibody or non-reactive were included. Reactive samples were characterized with FIDISTM Connective Profile (Theradiag) for CENP B, Jo-1, Ribosomal P, Scl-70, Sm, Sm/RNP, SS-A 60, TRIM21 (SS-A 52), SS-B and U1RNP; EliA CCP (Phadia) for CCP2, QUANTA Lite® Chromatin (Werfen) for Chromatin, QUANTA Flash® DFS70* (Werfen) for DFS70, Anti-dsDNA-IgG-ELISA (DRG) for dsDNA, and Immunodot (Euroimmun) for RNA polymerase III (RNAP III). For CCP2, only reactive samples were included. For the remaining analytes, non-reactive samples were characterized using immunofluorescence (ANA-Ro IgG FLUORESCENT HEp-2000®; Immuno Concepts, Germany) and ANAscreen ELISA (ORGENTEC-Diagnostika-GmbH, Germany). Each individual non-reactive sample was tested with the investigational device once; reactive samples were tested in duplicates. Positive percentage agreement (PPA) and negative percentage agreement (NPA) for individual analytes were calculated. For CCP2 only PPA is presented as no negative samples were included. Results Compared with relevant devices, the investigational prototype showed PPA ranging from 75% for chromatin to 99% for SS-A 60 and TRIM21. NPA ranged from 95% for chromatin, RNAP III, Scl-70 and Sm to 100% for DFS70, SS-A 60 and TRIM21. Performance details are shown in the Table. Conclusions The investigational prototype demonstrated substantial agreement with the compared CE-marked devices. Further studies will allow for expanded performance assessment of the investigational device. This fully-automated multiplexed platform has the potential to contribute to optimizing CTD evaluation by simplifying complex testing pathways and analyzing large number of samples per day.

B-072 Analytical Evaluation of a Novel Fully Automated Multiplexed Microarray Immunoassay for the Simultaneous Detection of Eleven Autoantibodies Associated with Connective Tissue Diseases in a Spanish Reference Laboratory

Abstract Background Detection of different autoantibodies is key in the identification of autoimmune diseases; however, most available devices are either individual tests and/or manual/semi-automated. Development of fully-automated multiplexed devices for autoantibody testing is needed. We evaluated the analytical performance of the novel MosaiQ® AiPlex CTD (AiPlex-CTD) microarray, used with the fully-automated MosaiQ system, for simultaneous qualitative detection of eleven autoantibodies associated with connective tissue diseases (CTD), compared with selected CE-marked devices. Methods AiPlex-CTD microarrays (AliveDx, Switzerland) were prepared by printing antigens onto functionalized glass chips. Microarrays consisting of 2 separate sides (1 side was printed, leaving the other side available for future addition of antigens) were assembled into magazines (containing 250 microarrays) for processing on the MosaiQ 125 instrument. Serum samples from a Spanish refence laboratory, characterized as reactive to ≥1 autoantibodies using Quanta Flash® CTD Screen (Werfen, Spain) or as non-reactive by FIDISTM Connective Profile (Theradiag, France). All samples were tested with AiPlex-CTD. Positive percent agreement (PPA) and negative percent agreement (NPA), overall and for individual analytes were calculated. Results Compared with Quanta Flash® CTD Screen, AiPlex-CTD showed PPA ranging from 80% for Sm to 100% for SS-A 60, TRIM21, U1RNP, Jo-1 and Scl-70. No reactive samples were available for Sm/RNP and Ribosomal P. Compared with FIDIS, NPA ranged from 95% for dsDNA, Scl-70 and CENP-B and 100% for SS-A 60, TRIM21, SS-B, Sm, Sm/RNP, U1RNP, Jo-1 and Ribosomal P. Performance details for individual analytes are shown in the Table. Conclusions In this sample cohort, AiPlex-CTD demonstrated high concordance with the compared CE-marked devices for the automated qualitative detection of the autoantibodies included in the assay, which is line with previous observations in a larger cohort using FIDIS and other CE-marked devices. This high throughput multiplexed platform has the potential to help optimize CTD testing by simplifying complex testing pathways.

B-094 Assessment of a Live Cell-based Flow Cytometry Assay for MuSK-IgG4 Testing in Myasthenia Gravis

Abstract Background Myasthenia gravis (MG) is caused by disrupting the function of postsynaptic acetylcholine receptors (AChRs) at the neuromuscular junction. 85% of MG patients have autoantibodies targeting AChRs, while 6% of MG patients have autoantibodies targeting the muscle-specific receptor tyrosine kinase (MuSK). MuSK is located on the muscle membrane and is activated by the low-density lipoprotein receptor-related protein 4 (LRP4) upon the binding with ligand Agrin. Activated MuSK undergoes autophosphorylation and stimulates downstream signaling to cluster AChRs, which is important for the formation and maintenance of neuromuscular synapses. IgG4 antibodies that bind MuSK are thought to directly disrupt the ability of MuSK to cluster AChR. The gold standard methodology for MuSK antibody testing is radioimmunoprecipitation assay (RIA) using anti-human IgG secondary antibodies. Performing RIA methods in the clinical laboratory is a burden due to radioactive biohazard concerns and the analytical limitations of these assays. This study is to assess the analytical and clinical performance of a live cell-based flow cytometry assay as a potential replacement for the MuSK RIA. Methods HEK293T cells were transiently transfected with a vector co-expressing human MuSK protein and GFP. Patient sera were incubated with a mix of HEK293 cells (GFP-positive cells that express MuSK protein on the cell surface and GFP-negative cells that do no express MuSK) followed by incubation with anti-human IgG4 secondary antibody conjugated with alexa fluor 647 (AF647). An IgG binding index (IBI) was calculated for each sample as the ratio of the median AF647 fluorescence intensity (MFI) of the MuSK-expressing cells (GFP+) to the MFI of the non-MuSK expressing cells (GFP-). An arbitrary positive IBI cut-off of 2.0 was selected. A preliminary assessment of assay imprecision was performed for the flow cytometry assay, as well as a method comparison study to compare this assay to RIA utilizing a cohort of 130 (34 positive and 96 negative) residual samples previously tested by the gold-standard assay. 133 disease control samples were also tested to assess the clinical specificity of the flow cytometry-based assay. Results To assess the imprecision, eight samples with varying IBIs were tested with 5 replicates in each assay over five days. The coefficient of variation (CV) for intra-assay replicates ranged from 1% to 7% for all samples, while CV for inter-assay replicates ranged from 2% to 15%. Based on the 130 samples previously tested by RIA, the Positive Percentage Agreement (PPA) between these two assays was 76.5% and the negative Percentage Agreement (NPA) was 100%. Of the discordant samples (n=8), clinical information was available for only 1 sample (positive by RIA, negative by flow). This patient had a final non-MG diagnosis (ALS). All control samples were negative on the flow cytometry-based assay. Conclusions The live cell-based flow cytometry MuSK IgG4 assay demonstrated encouraging preliminary analytical performance that warrants further development and validation.

Diagnostic Accuracy of the LabTurbo QuadAIO Common Flu Assay for Detecting Influenza A Virus, Influenza B Virus, RSV, and SARS-CoV-2.

Background: The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for rapid and accurate diagnostic tools for upper respiratory tract infections (URTIs). Nucleic acid amplification tests (NAATs) have transformed URTI diagnostics by enabling the rapid detection of multiple pathogens simultaneously, thereby improving patient management and infection control. This study aimed to evaluate the diagnostic accuracy of the LabTurbo QuadAIO Common Flu Assay compared to that of the Xpert Xpress CoV-2/Flu/RSV Plus Assay for detecting SARS-CoV-2, Influenza A, Influenza B, and respiratory syncytial virus (RSV). Methods: A retrospective diagnostic accuracy study was conducted using nasopharyngeal samples from patients. Samples were tested using the LabTurbo QuadAIO Common Flu Assay and the comparator Xpert Xpress CoV-2/Flu/RSV Plus Assay. Positive and negative percent agreements (PPA and NPA) were calculated. Results: The LabTurbo Assay demonstrated a PPA of 100% and an NPA of 100% for SARS-CoV-2, Influenza A, and Influenza B, whereas it showed a PPA of 100% and an NPA of 98.3% for RSV. Conclusions: The LabTurbo QuadAIO Assay exhibited high diagnostic accuracy for detecting multiple respiratory pathogens, including SARS-CoV-2, Influenza A, Influenza B, and RSV. Despite the slight discrepancy in the NPA for RSV, the overall performance of the LabTurbo Assay supports its integration into routine diagnostic workflows to enhance patient management and infection control.

A-267 How Does Rapid HIV Antibody Testing Fit Into the Current Testing Algorithm?

Abstract Background Many emergency departments (ED) currently use rapid HIV antibody tests as screening tools, despite limited sensitivity for detecting acute HIV infections. We performed an analysis of standard of care rapid and in-lab fourth generation HIV testing, to 1) assess the performance of rapid HIV testing in our patient population and 2) identify key symptoms of acute HIV infections. Methods A four-year retrospective analysis from 1/1/2018-12/31/2021 was conducted at Barnes Jewish Hospital and St. Louis Children’s Hospital in St. Louis, Missouri. During the study period, both rapid HIV testing (SURE CHECK HIV 1/2, Chembio) and in-lab fourth generation HIV testing (ARCHITECT HIV Ag/Ab Combo Assay, Abbott) were available to providers. Comparative performance of rapid testing and in-lab fourth generation testing was assessed using complete Fourth Generation HIV algorithmic testing as the gold standard. Symptomatology was recorded for a subset of patients that presented to the ED to determine associations with different testing patterns. Results A total of 28,240 rapid HIV antibody tests were administered throughout inpatient, outpatient, and emergency departments at both hospitals, revealing an average yearly positivity rate of 0.6%. In a subset of 1192 patients who underwent both rapid HIV antibody and fourth generation in-lab testing, the rapid HIV antibody test exhibited a positive percent agreement of 92.5% (95% CI: 84.6-96.5) and negative percent agreement of 98.1% (95% CI: 97.1-98.8). Rapid testing demonstrated a negative predictive value (NPV) of 99.5% (95% CI: 98.8-99.8) and a positive predictive value of 77.9% (95% CI: 68.6-85.1). Notably, the rapid test failed to detect 3/3 (100%) acute HIV infections defined as rapid HIV antibody test negative, Ag/Ab assay and nucleic acid amplification assay positive. The rapid test demonstrated comparable performance in patients with known HIV infections, regardless of antiretroviral therapy (ART) status. The positivity rate was 23/24 (95.8%) in known HIV patients with ART compared to 25/27 (92.6%) in those without (P&amp;gt;0.9999). Among patients on ART, the mean ± SD log10 viral load was 3.15±1.52, whereas those without ART exhibited a viral load of 4.87±0.99 (P=0.0140). In the ED, symptoms such as fever, nausea/vomiting, malaise, headache, and photophobia were significantly correlated with acute HIV infection. Acute HIV patients were more likely to display fever in conjunction with three other symptoms (P&amp;lt;0.0001). Conclusions The rapid HIV antibody test demonstrated high sensitivity, specificity, and NPV in our study population, reaffirming its effectiveness as a valuable screening tool. ART did not seem to affect the rapid test performance. However, the low PPV and 100% failure in detecting acute HIV infections underscore the importance of confirmatory in-lab testing. Insights into symptomatology of patients with acute HIV inform ED providers when confirmatory testing is essential for a timely and accurate HIV diagnosis.

Usability and acceptability of self-testing for hepatitis C virus exposure in a high-prevalence urban informal settlement in Karachi, Pakistan

BackgroundHepatitis C virus (HCV) antibody self-testing (HCVST) may help expand screening access and support HCV elimination efforts. Despite potential benefits, HCVST is not currently implemented in Pakistan. This study aimed to assess the usability and acceptability of HCVST in a high HCV prevalence informal settlement in Karachi, Pakistan.MethodsWe performed a cross-sectional study in a hepatitis C clinic from April through June 2023. Participants were invited to perform a saliva-based HCVST (OraSure Technologies, USA) while following pictorial instructions. A study member evaluated test performance using a standardized checklist and provided verbal support if a step could not be completed. Perceived usability and acceptability were assessed using a semi-structured questionnaire. The HCVST was considered successful if the participant was able to complete all steps and correctly interpret test results. Overall concordance and positive and negative agreement were estimated in comparison with the HCVST result read by the study member (inter-reader concordance and agreement) and result of a second rapid HCV test (Abbott Diagnostics Korea Inc, South Korea) performed by a trained user (inter-operator concordance and agreement).ResultsThe study included 295 participants of which 97 (32%) were illiterate. In total, 280 (95%, 95% CI 92–97%) HCVSTs were successful. Overall, 38 (13%) people performed the HCVST without verbal assistance, 67 (23%) needed verbal assistance in one step, 190 (64%) in two or more. Assistance was most often needed in managing the test buffer and test reading times. The inter-reader concordance was 96% and inter-operator concordance 93%. Inter-reader and inter-operator positive percent agreement were 84 and 70%, respectively. All participants reported they would use HCVST again and would recommend it to friends and family.ConclusionSaliva-based HCVST was very well accepted in this clinic-based setting. However, many people requested verbal support in several steps, highlighting the need for clear instructions for use and test devices that are simple to use, particularly in low literacy settings. Moderately low positive percent agreement with the results of a rapid test performed by a trained user highlights potential uncertainty in the accuracy of HCVST in the hands of lay users.

Analytical and Clinical Validation of the Oncomine Dx Target Test to Assess HER2 Mutation Status in Tumor Tissue Samples From Patients With Non–Small Cell Lung Cancer Treated With Trastuzumab Deruxtecan in the DESTINY-Lung01 and DESTINY-Lung02 Studies

Context.— Trastuzumab deruxtecan (T-DXd), a human epidermal growth factor receptor 2 (HER2)–targeted therapy, has demonstrated durable anticancer activity in patients with advanced, metastatic HER2 (also known as ERBB2)–mutant (HER2m) non–small cell lung cancer (NSCLC) who have limited treatment options and poor prognosis. Objective.— To analytically validate and assess the clinical utility of the Oncomine Dx Target (ODxT) Test as a companion diagnostic to identify patients with HER2m NSCLC. Design.— Tumor samples from patients in DESTINY-Lung01 and DESTINY-Lung02 were retrospectively analyzed alongside commercially procured samples using the ODxT test and compared to the assays used for screening in these clinical trials. Results.— Positive percent agreement (PPA) and negative percent agreement (NPA) between the ODxT Test and TruSight Tumor 170 assay when testing DESTINY-Lung01 and commercially procured samples met prespecified thresholds (PPA and NPA ≥90%) for analytical accuracy (100% and 99.1%). The ODxT Test results were highly concordant with clinical trial assays (CTAs) used in DESTINY-Lung01 (PPA and NPA, 98.0% and 100%) and DESTINY-Lung02 (PPA and NPA, 96.7% and 100%) to identify activating HER2 mutations in tumor samples. Confirmed objective response rates were similar between patients with HER2m tumors identified by the ODxT Test and by CTAs in DESTINY-Lung01 (58.3% and 54.9%) and DESTINY-Lung02 (53.6% and 53.8%). Response duration was 12.0 and 9.3 months for patients identified by the ODxT Test and CTAs, respectively, in DESTINY-Lung01. Conclusions.— The ODxT Test detected HER2 mutations in NSCLC with high analytical and clinical accuracy and identified HER2m populations with response rates similar to populations identified by CTAs, supporting clinical utility of the ODxT Test to inform treatment decisions for HER2m NSCLC.

Comparison of a dual antibody and antigen HCV immunoassay to standard of care algorithmic testing.

The Centers for Disease Control and Prevention (CDC) guidelines for hepatitis C virus (HCV) testing, although effective, may miss crucial diagnostic opportunities. The goal of this study was to assess the utility of an antibody (Ab) and antigen (Ag) combination immunoassay as an alternative to traditional HCV screening. Remnant specimens from 1,341 patients with concurrent third-generation serologic (Roche anti-HCV-II) and nucleic acid amplification testing (NAAT) were assessed using the HCV Duo Ab/Ag immunoassay (Roche). Patient demographics, risk factors, and standard of care (SOC) laboratory results from the medical records were recorded. Overall, 99.0% (197/199) of the HCV Duo Ab+/Ag+specimens accurately identified active infections as confirmed by NAAT, and 99.9% (670/671) Ab-/Ag- samples corresponded to those without HCV infections. Individually, the HCV Duo Ab component demonstrated a 95.6% positive percent agreement (PPA) (95% CI = 93.8-96.9) and 99.1% negative percent agreement (NPA) (98.8-99.6) compared with SOC anti-HCV II Ab assay. The HCV Duo Ag had a 73.5% PPA (67.9-78.4) and 99.8% NPA (99.3-100) with NAAT. Among RNA+ specimens, 73.4% (197/267) were HCV Duo Ag+, and 265/267 (99.3%) were successfully detected on the HCV Duo Ab component. Notably, 5/7 (71.4%) Ab-/RNA +specimens were detected by HCV Duo, which would have been missed by traditional algorithmic testing. Fourth generation HCV Duo Ab/Ag assay demonstrated comparable performance to SOC testing and shortens the diagnostic window but does not eliminate the need for NAAT in all patients. Ab/Ag testing identified several Ab-/RNA+ cases, a subgroup often undiagnosed by current algorithmic testing, demonstrating promise for improved diagnostic efficiency and accuracy in HCV detection.IMPORTANCEThis study highlights the potential of a combined hepatitis C virus (HCV) Duo antibody (Ab) and antigen (Ag) immunoassay to improve early detection of HCV infections. Traditional Ab-only screening methods recommended by the Centers for Disease Control and Prevention may miss early-stage infections. The HCV Duo assay showed high accuracy, detecting nearly all active infections confirmed by nucleic acid amplification testing. Dual detection of HCV Ab and Ag shortens the diagnostic window, enabling intervention and treatment in a single visit, which is crucial for improving patient outcomes and reducing HCV transmission, especially in areas with limited access to confirmatory molecular testing.

Benchmarking whole exome sequencing in the German network for personalized medicine

IntroductionWhole Exome Sequencing (WES) has emerged as an efficient tool in clinical cancer diagnostics to broaden the scope from panel-based diagnostics to screening of all genes and enabling robust determination of complex biomarkers in a single analysis. MethodsTo assess concordance, six formalin-fixed paraffin-embedded (FFPE) tissue specimens and four commercial reference standards were analyzed by WES as matched tumor-normal DNA at 21 NGS centers in Germany, each employing local wet-lab and bioinformatics. Somatic and germline variants, copy-number alterations (CNAs), and complex biomarkers were investigated. Somatic variant calling was performed in 494 diagnostically relevant cancer genes. The raw data were collected and re-analyzed with a central bioinformatic pipeline to separate wet- and dry-lab variability. ResultsThe mean positive percentage agreement (PPA) of somatic variant calling was 76 % while the positive predictive value (PPV) was 89 % in relation to a consensus list of variants found by at least five centers. Variant filtering was identified as the main cause for divergent variant calls. Adjusting filter criteria and re-analysis increased the PPA to 88 % for all and 97 % for the clinically relevant variants. CNA calls were concordant for 82 % of genomic regions. Homologous recombination deficiency (HRD), tumor mutational burden (TMB), and microsatellite instability (MSI) status were concordant for 94 %, 93 %, and 93 % of calls, respectively. Variability of CNAs and complex biomarkers did not decrease considerably after harmonization of the bioinformatic processing and was hence attributed mainly to wet-lab differences. ConclusionContinuous optimization of bioinformatic workflows and participating in round robin tests are recommended.

Comparing psychometric characteristics of a computerized cognitive test (BrainCheck Assess) against the Montreal cognitive assessment.

Previous validation studies demonstrated that BrainCheck Assess (BC-Assess), a computerized cognitive test battery, can reliably and sensitively distinguish individuals with different levels of cognitive impairment (i.e., normal cognition (NC), mild cognitive impairment (MCI), and dementia). Compared with other traditional paper-based cognitive screening instruments commonly used in clinical practice, the Montreal Cognitive Assessment (MoCA) is generally accepted to be among the most comprehensive and robust screening tools, with high sensitivity/specificity in distinguishing MCI from NC and dementia. In this study, we examined: (1) the linear relationship between BC-Assess and MoCA and their equivalent cut-off scores, and (2) the extent to which they agree on their impressions of an individual's cognitive status. A subset of participants (N = 55; age range 54-94, mean/SD = 80/9.5) from two previous studies who took both the MoCA and BC-Assess were included in this analysis. Linear regression was used to calculate equivalent cut-off scores for BC-Assess based on those originally recommended for the MoCA to differentiate MCI from NC (cut-off = 26), and dementia from MCI (cut-off = 19). Impression agreement between the two instruments were measured through overall agreement (OA), positive percent agreement (PPA), and negative percent agreement (NPA). A high Pearson correlation coefficient of 0.77 (CI = 0.63-0.86) was observed between the two scores. According to this relationship, MoCA cutoffs of 26 and 19 correspond to BC-Assess scores of 89.6 and 68.5, respectively. These scores are highly consistent with the currently recommended BC-Assess cutoffs (i.e., 85 and 70). The two instruments also show a high degree of agreement in their impressions based on their recommended cut-offs: (i) OA = 70.9%, PPA = 70.4%, NPA = 71.4% for differentiating dementia from MCI/NC; (ii) OA = 83.6%, PPA = 84.1%, NPA = 81.8% for differentiating dementia/MCI from NC. This study provides further validation of BC-Assess in a sample of older adults by showing its high correlation and agreement in impression with the widely used MoCA.

A Novel Next-Generation Sequencing Assay for the Identification of BCR::ABL1 Transcript Type and Accurate and Sensitive Detection of TKI-Resistant Mutations.

The clinical management of chronic myeloid leukemia (CML) patients requires the identification of the type of BCR::ABL1 transcript at diagnosis and the monitoring of its expression and potential tyrosine kinase inhibitor (TKI) resistance mutations during treatment. Detection of resistant mutation requires transcript type-specific amplification of BCR::ABL1 from RNA. In this study, a custom RNA-based next-generation sequencing (NGS) assay (Dup-Seq BCR::ABL1) that enables (a) the identification of BCR::ABL1 transcript type and (b) the detection of resistance mutations from common and atypical BCR::ABL1 transcript types was developed and validated. The assay design covers BCR exon 1 to ABL1 exon 10 and employs duplicate PCR amplification for error correction. The custom data analysis pipeline enables breakpoint determination and overlapped mutation calling from duplicates, which minimizes the low-level mutation artifacts. This study demonstrates that this novel assay achieves high accuracy (positive percent agreement (PPA) for fusion: 98.5%; PPA and negative percent agreement (NPA) for mutation at 97.8% and 100.0%, respectively) and sensitivity (limit of detection (LOD) for mutation detection at 3% from 10 000 copies of BCR::ABL1 input). The Dup-Seq BCR::ABL1 assay not only allows for the identification of BCR::ABL1 typical and atypical transcript types and accurate and sensitive detection of TKI-resistant mutations but also simplifies molecular testing work flow for the clinical management of CML patients.