High Positive Percent Agreement Research Articles

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 & 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.

Sample-to-answer direct real-time PCR detection of Anaplasma phagocytophilum, Ehrlichia spp., and Babesia spp. infections in whole-blood specimens.

This work demonstrates that detection of tick-borne illnesses, such as anaplasmosis, babesiosis, or ehrlichiosis, can be performed directly from whole blood with no extraction. The assay described here has a high positive and negative percent agreement with existing methods and is used as the standard of care. An increasing incidence of tick-borne illness combined with shortage of well-trained technologists to perform traditional manual testing, testing options that can be adapted to various lab settings, are of the utmost importance.

Development and evaluation of three automated media pooling and molecular diagnostic systems for the detection of SARS-CoV-2.

Pooled testing combined with molecular diagnostics for the detection of SARS-CoV-2 is a promising method that can increase testing capacities and save costs. However, pooled testing is also associated with the risks of decreased test sensitivity and specificity. To perform reliable pooled testing, we developed and validated three automated media pooling and molecular diagnostic systems. These pooling systems (geneLEAD-PS, Panther-PS, and Biomek-PS) comprised existing automated molecular detection platforms, corresponding automated media pooling devices, and laboratory information management systems. Analytical sensitivity analysis and mock sample evaluation were performed, and the obtained data were used to determine the sizes of the pool for the validation study. In the validation study, a total of 2,448, 3,228, and 6,420 upper respiratory samples were used for geneLEAD-PS, Panther-PS, and Biomek-PS, respectively, and the diagnostic performances were compared with the reference RT‒PCR assay. A pool size of 6 for geneLEAD-PS and a pool size of 4 for Panther-PS and Biomek-PS were selected for the validation studies. All three systems showed high positive percent agreement values of ≥90.5% and negative percent agreement values of ≥99.8% for any specimen type. Pooled testing resulted in a 65%-71% reduction in cost per sample. The testing capacities of geneLEAD-PS, Panther-PS, and Biomek-PS were 144 samples in 3 hours, 384 samples in 5.5 hours, and 376 samples in 4 hours, respectively. The developed pooling systems showed robust diagnostic performances and will increase the testing capacities of molecular diagnostic tests while saving costs and may contribute to infection control of COVID-19.IMPORTANCEDuring the COVID-19 pandemic, there have been surges in demand for accurate molecular diagnostic testing and laboratory supply shortages. Pooled testing combined with highly sensitive molecular testing, which entails mixing multiple samples as a single sample, is a promising approach to increase testing capacities while reducing the use of consumables. However, pooled testing is associated with risks that compromise diagnostic performance, such as false negatives due to dilution of positive samples or false positives due to cross-contamination. To perform reliable pooled testing, three different pooling systems (an automated pooling device, an automated molecular detection platform, and a laboratory information management system) were developed to accurately interpret pooled testing results. These three systems were validated using multiple clinical samples and showed high concordance with individual testing. The developed pooling systems will contribute to increasing reliable molecular testing capacities while using fewer consumables and saving costs.

Assessment of Bone Marrow Involvement in B-Cell non-Hodgkin Lymphoma Using Immunoglobulin Gene Rearrangement Analysis with Next-Generation Sequencing.

Assessment of bone marrow involvement (BMI) in non-Hodgkin lymphoma (NHL) is crucial for determining patient prognosis and treatment strategy. We assessed the prognostic value of next-generation sequencing (NGS)-based immunoglobulin (Ig) gene clonality analysis as an ancillary test for BMI evaluation in NHL. A retrospective cohort of 124 patients newly diagnosed with B-cell NHL between 2019 and 2022 was included. NGS-based Ig clonality analysis was conducted using LymphoTrak IGH FR1 Assay and IGK Assay (Invivoscribe Technologies, San Diego, CA, USA) on BM aspirate samples, and the results were compared with those of histopathological BMI (hBMI). Among the 124 patients, hBMI was detected in 16.9% (n = 21). The overall agreement of BMI between Ig clonality analyses and histopathological analysis for IGH, IGK, and either IGH or IGK was 86.3%, 92.7%, and 90.3%. The highest positive percent agreement was observed with clonal rearrangements of either IGH or IGK gene (90.5%), while the highest negative percent agreement was observed with clonal rearrangement of IGK gene (96.1%). For the prediction of hBMI, positive prediction value ranged between 59.1% and 80.0% and the negative prediction value ranged between 91.3% and 97.9%. NGS-based clonality analysis is an analytic platform with a substantial overall agreement with histopathological analysis. Assessment of both IGH and IGK genes for the clonal rearrangement analysis could be considered for the optimal diagnostic performance of BMI detection in B-cell NHL.

Applicability of Bronchoalveolar Lavage Fluid and Plasma Metagenomic Next-Generation Sequencing Assays in the Diagnosis of Pneumonia.

Metagenomic next-generation sequencing (mNGS) provides innovative solutions for predicting complex infections. A comprehensive understanding of its strengths and limitations in real-world clinical settings is necessary to ensure that it is not overused or misinterpreted. Two hundred nine cases with suspected pneumonia were recruited to compare the capabilities of 2 available mNGS assays (bronchoalveolar lavage fluid [BALF] mNGS and plasma mNGS) to identify pneumonia-associated DNA/RNA pathogens and predict antibiotic resistance. Compared to clinical diagnosis, BALF mNGS demonstrated a high positive percent agreement (95.3%) but a low negative percent agreement (63.1%). Plasma mNGS revealed a low proportion of true negatives (30%) in predicting pulmonary infection. BALF mNGS independently diagnosed 65.6% (61/93) of coinfections and had a remarkable advantage in detecting caustic, rare, or atypical pathogens. Pathogens susceptible to invasive infection or bloodstream transmission, such as Aspergillus spp, Rhizopus spp, Chlamydia psittaci, and human herpesviruses, are prone to be detected by plasma mNGS. BALF mNGS tests provided a positive impact on the diagnosis and treatment of 128 (61.2%) patients. Plasma mNGS, on the other hand, turned out to be more suitable for diagnosing patients who received mechanical ventilation, developed severe pneumonia, or developed sepsis (all P < .01). BALF mNGS was able to identify resistance genes that matched the phenotypic resistance of 69.4% (25/36) of multidrug-resistant pathogens. Our data reveal new insights into the advantages and disadvantages of 2 different sequencing modalities in pathogen identification and antibiotic resistance prediction for patients with suspected pneumonia.

Diagnostic utility of oropharyngeal swabs as an alternative to lower respiratory tract samples for PCR-based syndromic testing in patients with community-acquired pneumonia.

Syndromic PCR-based analysis of lower respiratory tract (LRT) samples in patients with community-acquired pneumonia (CAP) improves the bacterial yield and time-to-results compared to culture-based methods. However, obtaining adequate sputum samples can be challenging and is frequently not prioritized in the emergency department (ED). In this study, we assess the concordance of microbiological detections between oropharyngeal- (OP) and LRT samples from patients presenting to the ED with CAP using a syndromic PCR-based respiratory panel [Biofire FilmArray Pneumonia plus (FAP plus)]. Paired OP- and high-quality LRT samples were collected from 103 patients with confirmed CAP, who had been included in a randomized controlled trial (NCT04660084) or a subsequent observational study at Haukeland University Hospital, and analyzed using the FAP plus. The LRT samples were obtained mainly by sputum induction (88%). Using the LRT samples as a reference standard, the positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement for the most common bacterial pathogens in CAP, Streptococcus pneumoniae and Haemophilus influenzae, were 85%, 99% and 95%, and 86%, 98% and 93%, respectively. For Moraxella catarrhalis, the PPA was lower (74%), while the NPA was 100%. For bacteria that are less likely causes of uncomplicated CAP (e.g., Staphylococcus aureus and Enterobacterales) the results were more divergent. In conclusion, the FAP plus detects the most common CAP pathogens S. pneumoniae and H. influenzae from OP samples with high PPAs and excellent NPAs when compared with LRT samples. For these pathogens, the PPAs for OP samples were higher than previous reports for nasopharyngeal samples. This suggests that analysis of OP samples with syndromic PCR panels could represent an alternative approach for rapid microbiological testing in the ED, especially in patients where LRT samples are difficult to obtain. Divergent results for bacteria that are less likely to cause uncomplicated CAP do, however, emphasize the need for clinical evaluation of positive test results.

Clinical assessment of HRD scoring in relation to PARPi response in terms of statistical guidance on reporting results for CDx tests evaluation.

e17597 Background: Homologous recombination deficiency (HRD) is a tumor characteristic that can be assessed via different biomarkers such as individual mutations in BRCA1 and BRCA2 genes, and/or other HRR genes and genomic instability. We assessed a combined HRD score in three measures; BRCAs mutations, HRR mutation (HRRm) status, and genomic instability scoring via a specific bioinformatic algorithm of the testing panel to establish clinical validation. Methods: We tested 157 ovarian cancer patients’ FFPE samples to define HRD score, BRCA1/2 mutation and other HRRm status, to camper the responses to PARP inhibitors. For the HRD scoring, all the samples that were previously tested with alternative HRD tests and the cases had an indication of PARPi also tested via HRDInfo (CE-IVD) Panel for clinical diagnostics in this study. The PPA, NPA and OPA have been measured according to the FDA regulations of overall agreement for a follow-on CDx to show clinical evidence. Results: Depending on the three levels of testing strategy, BRCA1-2 mutation status in relation to the clinical response for PARPi has the lowest positive percent agreement (PPA) with 63.41% while HRD score has the highest with 97.62% that is higher than the any alternative HRD tests. All the PPA, NPA and OPA measures showed in the Figure for BRCA1-2 status and Genomic Instability stand alone and all BRCA1-2 plus other HRR genes plus Genomic Instability Status (GIS) to define HRD scoring. Conclusions: Even though the testing rate of HRD tests are still low all around the world and limited to specific cancers, there is an increasing requirement for different types of cancers. However, most alternative HRD tests have high overall concordance but fail to reach FDA CDx test requirements. Thus, this study showing the clinical evidence in the treatment of PARPi response data together with high PPA, NPA and OPA measures of HRDInfo panel and its bioinformatics algorithm. [Table: see text]

Abstract 3135: Validation of a bioinformatic model for classifying non-tumor variants in a cell-free DNA liquid biopsy assay

Abstract Background: Liquid biopsy has been established as a powerful, non-invasive means to profile tumors in and identify clinically relevant variants. However, the presence of clonal hematopoiesis (CH) variants, or biological noise, due to aging or therapy has potential to confound biomarker interpretation. Currently, comprehensive methods to filter out non-tumor variants require genotyping the white blood cell (WBC) fraction of the paired plasma sample, which is a costly, complicated workflow. A plasma-only, bioinformatics solution to identify non-tumor variants is needed for accurate biomarker assessments in the cell-free DNA (cfDNA). Method: An ensemble model was trained on a database of &amp;gt;250,000 plasma samples comprising healthy donor, early and late-stage cancer patients sequenced on the Guardant360TM , GuardantREVEALTM, and GuardantOMNITM liquid biopsy panels as well as public tissue datasets. The model was optimized with 5 fold cross-validation and hyperparameter tuning to produce a non-tumor and tumor variant classifier. To validate these calls, 116 paired plasma and WBC advanced cancer samples were selected for high prevalence of putative CH variants and genotyped using an in-house bioinformatics pipeline. In the validation cohort, cfDNA variants were determined to be of non-tumor or CH origin if there was molecule support in the WBC; cfDNA variants above 0.6% (limit of detection in the gDNA) with no support in the WBC were determined to be from the tumor. Results: The validation cohort consisted of 2150 somatic SNV and Indels, 956 of which were confirmed in the WBC and 1194 confirmed as plasma-only. Half of confirmed CH variants (48%, 458/956) occurred in known CH genes, while the other half occurred in genes such as TP53, ATM, NOTCH4, FAT1, SRSF2. No clinically actionable variants were confirmed in the WBC. Non-tumor or CH predictions were made for 624 somatic variants: 515/624 correctly identified CH, for a positive predictive value (PPV) of 83%. Of all CH variants confirmed in the WBC, 54% (553/956) had a CH or non-CH prediction; CH predictions had 91% (515/553) positive percent agreement (PPA) with the WBC. Remaining variants with no CH prediction (403/956) were low or no prevalence across datasets and occurred predominantly in LRP1B, TET2, TP53, KMT2D. Nearly half (67%, n=109) of CH predictions not in WBC occurred in a CH gene. For non-CH gene variants, 16% of false positive predictions occurred in 6 variants across 4 genes (ACVR2A, RNF43, B2M, FLT3). Conclusion: We present a plasma-only method that has high PPA and PPV with WBC genotyping for classifying non-tumor, CH variants in the cfDNA. Further investigation is underway to improve the sensitivity of annotating rare CH variants. Accurate CH identification is critical for treatment selection across targeted therapies, particularly for loss of function variants in DNA repair genes that may confer sensitivity to PARPi or ATRi therapies. Citation Format: Jennifer Yen, Yu Fu, Jeff Werbin, Katie Quinn, Robert Foley, Minh Trahn, Carin Espenschied, Scott Higdon, Brett Kennedy, Han-Yu Chuang. Validation of a bioinformatic model for classifying non-tumor variants in a cell-free DNA liquid biopsy assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3135.

Performance of VIDAS® Diagnostic Tests for the Automated Detection of Dengue Virus NS1 Antigen and of Anti-Dengue Virus IgM and IgG Antibodies: A Multicentre, International Study

Dengue is a serious mosquito-transmitted disease caused by the dengue virus (DENV). Rapid and reliable diagnosis of DENV infection is urgently needed in dengue-endemic regions. We describe here the performance evaluation of the CE-marked VIDAS® dengue immunoassays developed for the automated detection of DENV NS1 antigen and anti-DENV IgM and IgG antibodies. A multicenter concordance study was conducted in 1296 patients from dengue-endemic regions in Asia, Latin America, and Africa. VIDAS® dengue results were compared to those of competitor enzyme-linked immunosorbent assays (ELISA). The VIDAS® dengue assays showed high precision (CV ≤ 10.7%) and limited cross-reactivity (≤15.4%) with other infections. VIDAS® DENGUE NS1 Ag showed high positive and negative percent agreement (92.8% PPA and 91.7% NPA) in acute patients within 0-5 days of symptom onset. VIDAS® Anti-DENGUE IgM and IgG showed a moderate-to-high concordance with ELISA (74.8% to 90.6%) in post-acute and recovery patients. PPA was further improved in combined VIDAS® NS1/IgM (96.4% in 0-5 days acute patients) and IgM/IgG (91.9% in post-acute patients) tests. Altogether, the VIDAS® dengue NS1, IgM, and IgG assays performed well, either alone or in combination, and should be suitable for the accurate diagnosis of DENV infection in dengue-endemic regions.

Analytical and clinical performances of seven direct detection assays for SARS-CoV-2

BackgroundDirect detection tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that bypass complicated nucleic acid/antigen purification steps are promising tools for the rapid diagnosis of coronavirus disease 2019 (COVID-19). MethodsTo determine the analytical and clinical diagnostic performances of the direct detection assays, we compared 6 direct molecular detection assays, including two loop-mediated isothermal amplification (LAMP) assays and one lateral flow antigen assay, against the reference extraction-based RT-PCR assay using 183 respiratory samples (87 nasopharyngeal swabs, 51 saliva samples, and 45 sputum samples). ResultsAnalytical sensitivity analysis showed that the direct RT-PCR assay of Toyobo exhibited the lowest LOD of 1,000 copies/mL. Compared with the 80 positive and 103 negative samples based on the reference assay, the Toyobo assay had the highest positive percent agreement (PPA) of 96.3%, followed by the two direct RT-PCR assays of Takara and Shimadzu and one LAMP assay of Eiken (86.3–87.5%). The Fujirebio antigen assay had the lowest PPA of 44.7% among the assays tested. The negative percent agreement of these direct detection assays was 100%, except for the Eiken assay (96.3%). ConclusionsLarge differences in PPA existed among the direct detection tests. Laboratories need to take these characteristics into consideration before implementing these assays.

Comparative analysis of three multiplex platforms for the detection of respiratory viral pathogens.

Acute viral respiratory infections are a major health burden in children worldwide. In recent years, rapid and sensitive multiplex nucleic acid amplification tests (NAATs) have replaced conventional methods for routine virus detection in the clinical laboratory. We compared BioFire® FilmArray® Respiratory Panel (FilmArray V1.7), Luminex NxTag® Respiratory Pathogen Panel (NxTag RPP) and Applied Biosystems TaqMan Array Card (TAC) for the detection of eight viruses in pediatric respiratory specimens. Results from the three platforms were analyzed with a single-plex real-time RT-PCR (rRT-PCR) assay for each virus. Of the 170/210 single-plex virus-positive samples, FilmArray detected a virus in 166 (97.6%), TAC in 163 (95.8%) and NxTag RPP in 160 (94.1%) samples. The Positive Percent Agreement (PPA) of FilmArray, NxTag RPP and TAC was highest for influenza B (100%, 100% and 95.2% respectively) and lowest for seasonal coronaviruses on both FilmArray (90.2%) and NxTag RPP (81.8%), and for parainfluenza viruses 1- 4 on TAC (84%). The Negative Percent Agreement (NPA) was lowest for rhinovirus/enterovirus (92.9%, 96.7% and 97.3%) on FilmArray, NxTag RPP and TAC respectively. NPA for all three platforms was highest (100%) for both parainfluenza viruses 1- 4 and influenza A and B, and 100% for human metapneumovirus with TAC as well. All three multiplex platforms displayed high overall agreement (&gt;90%) and high NPA (&gt;90%), while PPA was pathogen dependent and varied among platforms; high PPA (&gt;90%) was observed for FilmArray for all eight viruses, TAC for six viruses and NxTag RPP for 4 viruses.

Novel CRISPR-based detection of Leishmania species.

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10−2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

Targeted Versus Shotgun Metagenomic Sequencing-based Detection of Microorganisms in Sonicate Fluid for Periprosthetic Joint Infection Diagnosis.

Next-generation sequencing (NGS) is increasingly used for periprosthetic joint infection (PJI) diagnosis, but its clinical utility is poorly defined. Shotgun metagenomic sequencing (sNGS) has been reported to identify PJI pathogens undetected by culture in sonicate fluid. However, sNGS is complex and costly. Here, 16S ribosomal RNA (rRNA) gene-based targeted metagenomic sequencing (tNGS) was compared to sNGS of sonicate fluid for microbial detection and identification in patients with total hip arthroplasty (THA) and total knee arthroplasty (TKA) failure. A convenience sample of sonicate fluids derived from patients who had undergone THA or TKA removal, enriched with culture negative PJI cases, was tested. Samples had been previously tested by sNGS. For tNGS, samples were extracted, amplified by polymerase chain reaction targeting the V1 to V3 regions of the 16S rRNA gene, and sequenced on an Illumina MiSeq. A total of 395 sonicate fluids, including 208 from subjects with PJI, were studied. Compared with sonicate fluid culture, tNGS had higher positive percent agreement (72.1 vs 52.9%, P < .001), detecting potential pathogens in 48.0% of culture-negative PJIs. There was no difference between the positive percent agreement of tNGS (72.1%) and sNGS (73.1%, P = .83). 16S rRNA gene-based tNGS is a potential diagnostic tool for PJI pathogen identification in sonicate fluid from failed THAs and TKAs in culture-negative cases, with similar performance characteristics to sNGS.

Cepheid Xpert Xpress Flu/RSV evaluation performed by minimally trained non-laboratory operators in a CLIA-waived environment

The COVID-19 pandemic highlighted the significance of readily available and easily performed viral testing for surveillance during future infectious pandemics. The objectives of this study were: to assess the performance of the Xpert Xpress Flu and/or RSV test, a multiplex PCR assay for detecting influenza A and B virus and respiratory syncytial virus nucleic acids in respiratory tract specimens, relative to the Quidel Lyra Influenza A+B assay and the Prodesse ProFlu+ assay, and the system's ease of use by minimally trained operators. Overall, the Xpert Xpress Flu/RSV test demonstrated a high positive and negative percent agreement with the comparator assays, and was easy to use and interpret results, based on the operators’ feedback. We concluded that the Xpert Xpress Flu/RSV test is sensitive, specific, and easy to use for the diagnosis of influenza and RSV by minimally trained operators and can be a valuable tool in future infectious clusters or pandemics.

A Method for Variant Agnostic Detection of SARS-CoV-2, Rapid Monitoring of Circulating Variants, and Early Detection of Emergent Variants Such as Omicron.

ABSTRACTThe rapid emergence of SARS-CoV-2 variants raised public health questions concerning the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to understand transmission patterns and loads on healthcare resources. Next-generation sequencing (NGS) is the primary method for detecting and tracing new variants, but it is expensive, and it can take weeks before sequence data are available in public repositories. This article describes a customizable reverse transcription PCR (RT-PCR)-based genotyping approach which is significantly less expensive, accelerates reporting, and can be implemented in any lab that performs RT-PCR. Specific single-nucleotide polymorphisms (SNPs) and indels were identified which had high positive-percent agreement (PPA) and negative-percent agreement (NPA) compared to NGS for the major genotypes that circulated through September 11, 2021. Using a 48-marker panel, testing on 1,031 retrospective SARS-CoV-2 positive samples yielded a PPA and NPA ranging from 96.3 to 100% and 99.2 to 100%, respectively, for the top 10 most prevalent World Health Organization (WHO) lineages during that time. The effect of reducing the quantity of panel markers was explored, and a 16-marker panel was determined to be nearly as effective as the 48-marker panel at lineage assignment. Responding to the emergence of Omicron, a genotyping panel was developed which distinguishes Delta and Omicron using four highly specific SNPs. The results demonstrate the utility of the condensed panel to rapidly track the growing prevalence of Omicron across the US in December 2021 and January 2022.

Clinical Validation of a Novel T-Cell Receptor Sequencing Assay for Identification of Recent or Prior Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

BackgroundWhile diagnostic, therapeutic, and vaccine development in the COVID-19 pandemic has proceeded at unprecedented speed, critical gaps in our understanding of the immune response to SARS-CoV-2 remain unaddressed by current diagnostic strategies.MethodsA statistical classifier for identifying prior SARS-CoV-2 infection was trained using >4000 SARS-CoV-2–associated TCRβ sequences identified by comparing 784 cases and 2447 controls from 5 independent cohorts. The T-Detect™ COVID assay applies this classifier to TCR repertoires sequenced from blood samples to yield a binary assessment of past infection. Assay performance was assessed in 2 retrospective (n = 346; n = 69) and 1 prospective cohort (n = 87) to determine positive percent agreement (PPA) and negative percent agreement (NPA). PPA was compared to 2 commercial serology assays, and pathogen cross-reactivity was evaluated.ResultsT-Detect COVID demonstrated high PPA in individuals with prior RT-PCR–confirmed SARS-CoV-2 infection (97.1% 15 + days from diagnosis; 94.5% 15 + days from symptom onset), high NPA (∼100%) in presumed or confirmed SARS-CoV-2 negative cases, equivalent or higher PPA than 2 commercial serology tests, and no evidence of pathogen cross-reactivity.ConclusionT-Detect COVID is a novel T-cell immunosequencing assay demonstrating high clinical performance for identification of recent or prior SARS-CoV-2 infection from blood samples, with implications for clinical management, risk stratification, surveillance, and understanding protective immunity and long-term sequelae.

Clinical Performance of a Lateral Flow SARS-CoV-2 Total Antibody Assay.

BackgroundSerological assays for SARS-CoV-2 are important tools for diagnosis in patients with negative RT-PCR testing, pediatric patients with multisystem inflammatory syndrome, and serosurveillance studies. However, lateral flow-based serological assays have previously demonstrated poor analytical and clinical performance, limiting their utility.MethodsWe assessed the ADEXUSDx COVID-19 lateral flow assay for agreement with diagnostic RT-PCR testing using 120 specimens from RT-PCR–positive patients, 77 specimens from symptomatic RT-PCR–negative patients, and 47 specimens obtained prepandemic. Specimens collected <14 days from symptom onset in RT-PCR–positive patients were compared relative to the Abbott SARS-CoV-2 IgG assay.ResultsThe ADEXUSDx COVID-19 Test yielded an overall positive percent agreement (PPA) of 92.5% (95%CI 85.8 to 96.3) and negative percent agreement of 99.2% (95% CI 94.9–100.0) relative to RT-PCR and in prepandemic specimens. Relative to days from symptom onset, the PPA after 13 days was 100% (95% CI 94.2–100); from 7 to 13 days, 89.7 (95% CI 71.5–97.2); and from 0 to 7 days, 53.8 (95% CI 26.1–79.6). The overall agreement between the Abbott and ADEXUSDx assays was 80.9%. Twenty-five specimens were positive by both assays, 9 specimens were negative by both assays, and 8 specimens were positive by only the ADEXUSDx assay.ConclusionsWe demonstrate high PPA and negative percent agreement of the ADEXUSDx COVID-19 assay and diagnostic testing by RT-PCR, with PPA approximately 90% by 7 days following symptom onset. The use of waived testing for antibodies to SARS-CoV-2 with high sensitivity and specificity provide a further tool for combatting the COVID-19 pandemic.

Qualitative and quantitative detection of SARS-CoV-2 antibodies from dried blood spots

IntroductionDried blood spot (DBS) sampling is a minimally invasive method for specimen collection with potential multifaceted uses, particularly for serosurveillance of previous SARS-CoV-2 infection. In this study, we assessed DBS as a potential specimen type for assessing IgG and total (including IgG and IgM) antibodies to SARS-CoV-2 in vaccinated and naturally infected patients. MethodsSix candidate buffers were assessed for eluting blood from DBS cards. The study utilized one hundred and five paired plasma specimens and DBS specimens from prospectively collected SARS-CoV-2 vaccinated individuals, remnants from those with PCR confirmed SARS-CoV-2 infections, or remnants from those without history of infection or vaccination. All specimens were tested with the Siemens SARS-CoV-2 total assay (COV2T) or IgG assay (sCOVG). ResultsThe lowest backgrounds were observed with water and PBS, and water was used for elution. Relative to plasma samples, DBS samples had a positive percent agreement (PPA) of 94.4% (95% CI: 94.9–100%) for COV2T and 79.2 (68.4–87.0) for sCOVG using the manufacturer’s cutoff. The NPA was 100 % (87.1–100.0 and 85.13–100) for both assays. Dilution studies revealed 100% (95% CI: 90.8–100%) qualitative agreement between specimen types on the COV2T assay and 98.0% (88.0–99.9%) with the sCOVG using study defined cutoffs. ConclusionDBS specimens demonstrated high PPA and NPA relative to plasma for SARS-CoV-2 serological testing. Our data support feasibility of DBS sampling for SARS-CoV-2 serological testing.

144. Clinical Validation and Performance of a T-cell Immunosequencing Assay to Identify Past SARS-CoV-2 Infection

BackgroundOur understanding of the SARS-CoV-2 immune response has critical gaps that are inadequately addressed with available tools. We report the clinical performance of T-Detect COVID, the first T-cell assay to identify prior SARS-CoV-2 infection using T-cell receptor (TCR) sequencing and repertoire profiling from whole blood samples. MethodsThe T-Detect COVID assay combines high-throughput immunosequencing of the TCRß gene from blood samples with a statistical classifier demonstrating 99.8% specificity for identifying prior SARS-CoV-2 infection. The assay was employed in several retrospective and prospective cohorts to assess primary and secondary Positive Percent Agreement (PPA) with SARS-CoV-2 RT-PCR (N=205; N=77); primary and secondary Negative Percent Agreement (NPA; N=87; N=79); PPA compared to SARS-CoV-2 serology (N=55); and pathogen cross-reactivity (N=38). The real-world performance of the test was also evaluated in a retrospective review of test ordering (N=69) at a single primary care clinic in Park City, Utah. ResultsIn validation studies, T-Detect COVID demonstrated high PPA (97.1% ≥15 days from diagnosis) in subjects with prior PCR-confirmed SARS-CoV-2 infection; high NPA (~100%) in SARS-CoV-2 negative cases; equivalent or higher PPA with RT-PCR compared to two commercial EUA antibody tests; and no evidence of pathogen cross-reactivity. Review of assay use in a single clinic showed 100% PPA with RT-PCR in individuals with past confirmed SARS-CoV-2 vs. 85.7% for antibody testing, 100% agreement with positive antibody results, and positive results in 2/4 convalescent subjects with seroreversion to a negative antibody. In addition, 12/69 (17.3%) individuals with absent or negative RT-PCR tested positive by T-Detect COVID, nearly all of whom had compatible symptoms and/or exposure. TCR positivity was observed up to 12+ months (median 118 days) from the date of positive RT-PCR. ConclusionA T-cell immunosequencing assay shows high clinical performance for identifying past SARS-CoV-2 infection from whole blood samples. This assay can provide additional insights on the SARS-CoV-2 immune response, with practical implications for clinical management, risk stratification, surveillance, assessing vaccine immunity, and understanding long-term sequelae. Disclosures Sudeb C. Dalai, MD, PhD, Adaptive Biotechnologies (Employee, Shareholder) Jennifer N. Dines, MD, Adaptive Biotechnologies (Employee, Shareholder) Thomas M. Snyder, PhD, Adaptive Biotechnologies (Employee, Shareholder) Rachel M. Gittelman, PhD, Adaptive Biotechnologies (Employee, Shareholder) Tera Eerkes, PhD, Adaptive Biotechnologies (Employee, Shareholder) Pashmi Vaney, PhD, Adaptive Biotechnologies (Employee, Shareholder) Sally Howard, PhD, Adaptive Biotechnologies (Employee, Shareholder) Kipp Akers, PhD, Adaptive Biotechnologies (Employee, Shareholder) Lynell Skewis, PhD, Adaptive Biotechnologies (Employee, Shareholder) Anthony Monteforte, PhD, Adaptive Biotechnologies (Employee, Shareholder) Pamela R. Witte, PhD, Adaptive Biotechnologies (Employee, Shareholder) Cristina Wolf, PhD, Adaptive Biotechnologies (Employee, Shareholder) Hans Nesse, PhD, Adaptive Biotechnologies (Employee, Shareholder) Jia Qadeer, PhD, Adaptive Biotechnologies (Employee, Shareholder) Sarah Duffy, PhD, Adaptive Biotechnologies (Employee, Shareholder) Emily Svejnoha, PhD, Adaptive Biotechnologies (Employee, Shareholder) Caroline Taromino, PhD, Adaptive Biotechnologies (Employee, Shareholder) Ian M. Kaplan, PhD, Adaptive Biotechnologies (Employee, Shareholder) John Alsobrook, MD, Adaptive Biotechnologies (Employee, Shareholder) Thomas Manley, MD, Adaptive Biotechnologies (Employee, Shareholder) Lance Baldo, MD, Adaptive Biotechnologies (Employee, Shareholder, Leadership Interest)

Refractive outcomes of table-mounted and hand-held auto-refractometers in children: an observational cross-sectional study

BackgroundTo compare the refractive results of hand-held and table-mounted autorefractors.MethodsWe designed this study as an observational, cross-sectional study. We compared the mean spheric and cylinder power, spherical equivalent, Jackson cross-cylinder values, determined the limits of agreement (LoA), and evaluated the reliability of two autorefractors.ResultsWe evaluated 256 eyes of 256 pediatric patients (mean age, 9.12 ± 2.26 years; range, 5–16 years). 49% of the patients were female, and 51% were male. The Nidek HandyRef-K autorefractor measured relatively more astigmatism (P < 0.001) and less hyperopia (P = 0.024). The mean differences and 95% LoA were 0.06 D ± 0.47 D (− 0.82 D to 0.98 D) in spherical power, 0.08 D ± 0.28 D (− 0.47 D to 0.64 D) in cylindrical power, 0.11 D ± 0.47 D (− 0.81 D to 1.01 D) in spherical equivalent, 0.02 D ± 0.36 D (− 0.73 D to 0.69 D) in Jackson cross-cylinder power at 0°, 0.005 D ± 0.54 D (− 1.07 D to 1.06 D) in Jackson cross-cylinder power at 45°. We found the difference within 0.50 D in 244 (95%) eyes for spherical power, in 245 (96%) eyes for cylindrical power, 228 (89%) eyes for spherical equivalent, 224 (87%) eyes for Jackson cross-cylinder power at 0°, 213 (83%) eyes for Jackson cross-cylinder power at 45°. When comparing devices, there were strong correlations for spherical power (Spearman’s rho = 0.99, P < 0.001), cylindrical power (Spearman’s rho = 0.88, P < 0.001), and spherical equivalent (Spearman’s rho = 0.98, P < 0.001).ConclusionTwo autorefractors showed clinically applicable agreement limits; excellent reliability for spherical power and spherical equivalent and good reliability for cylindrical power; high positive percent agreement for spherical and cylindrical power, spherical equivalent, Jackson cross-cylinder power at 0°and 45°. These results showed that both devices might be used interchangeably for screening of refractive error in children.