Negative Percent Agreement Research Articles

A rapid, multiplex digital PCR assay to detect gene variants and fusions in non-small cell lung cancer.

Digital PCR (dPCR) is emerging as an ideal platform for the detection and tracking of genomic variants in cancer due to its high sensitivity and simple workflow. The growing number of clinically actionable cancer biomarkers creates a need for fast, accessible methods that allow for dense information content and high accuracy. Here, we describe a proof-of-concept amplitude modulation-based multiplex dPCR assay capable of detecting 12 single-nucleotide and insertion/deletion (indel) variants in EGFR, KRAS, BRAF, and ERBB2, 14 gene fusions in ALK, RET, ROS1 and NTRK1, and MET exon 14 skipping present in non-small cell lung cancer (NSCLC). We also demonstrate the use of multi-spectral target-signal encoding to improve the specificity of variant detection by reducing background noise by up to an order of magnitude. The assay reported an overall 100% positive percent agreement (PPA) and 98.5% negative percent agreement (NPA) compared to a sequencing-based assay in a cohort of 62 human formalin-fixed paraffin-embedded (FFPE) samples. In addition, the dPCR assay rescued actionable information in ten samples that failed to sequence, highlighting the utility of a multiplexed digital PCR assay as a potential reflex solution for challenging NSCLC samples.

Evaluation of an Automated Executed Version of an ELISA for the Serological Diagnosis of Dengue Infections

The incidence of dengue is increasing in many countries and at the present time, around half of the world's population is at risk of infection. Dengue is an arthropod-borne virus (arbovirus) that continues to pose an important challenge to public health authorities worldwide. A critical task in managing arboviral outbreaks and epidemics is the selection of laboratory technology for timely and accurate diagnosis. In this study, we assessed the performance of the DYNEX® DS2 Automated Processing System for the diagnosis of dengue vis a vis the manually executed procedure. The levels of agreement were calculated and showed 94%, 96%, and 90% positive percent agreement, negative percent agreement, and overall percent agreement, respectively. In conclusion, both tests displayed comparable performances.

Rapid and automated screening of carbapenemase- and ESBL-producing Gram-negative bacteria from rectal swabs using chromogenic agar media and the ScanStation device.

The ScanSation 100 device (Interscience, France) is an incubator allowing real-time detection of bacterial colony growth by frequently imaging agar plates over time, counting CFU, and detecting colony color. This study evaluated its performance for the early detection of carbapenemase-producing bacteria (CPB) and extended-spectrum β-Lactamase-producing bacteria (ESBL-PB) from rectal swabs inoculated on CHROMagar mSuperCARBA and ESBL media, respectively. Rectal screening ESwabs collected from patients admitted to Grenoble University Hospital between January and June 2021 were analyzed. After inoculation, chromogenic media were incubated for 24 h in the automaton, with image acquisition every 30 min. ScanStation results were compared to visual observations of the plates after 24 h of incubation. In total, 501 rectal swabs were tested. ScanStation showed 100% positive percent agreement (PPA) for the detection of CPB and ESBL-PB, whereas the PPA of color categorization ranged between 45% and 100%. Negative percent agreement (NPA) ranged between 70% and 98%. Negative predictive values (NPVs) were 100% for both bacterial groups, whereas positive predictive values (PPVs) were 70.3% for CPB and 81.0% for ESBL-PB. Importantly, real-time screening allowed detection of the first suspected colony within 10-14 h of growth, on average, whereas visual observation is usually only performed once a day after 18-24 h of incubation. Our study demonstrates the potential use of early images to accelerate the detection of CPB and ESBL-PB and implement effective and timely infection control measures. IMPORTANCE The ScanStation 100 device is an incubator able to follow the real-time growth of bacterial colonies on agar plates through digital imaging, allowing users to sort plates according to the presence or absence of colonies, and to distinguish their color using four numeric color filters. Real-time screening shows that first colony detection is possible much earlier (after 10-14 h of growth, on average), whereas visual observation is usually performed only once a day after 18-24 h of incubation. The ScanStation device, combined with chromogenic agar media, is an efficient automated screening method to accelerate the detection of Gram-negative multidrug-resistant bacteria in laboratories that do not have access to larger laboratory automation systems. Our study shows that setting the image acquisition to one or two early images may allow for the detection of positive samples that were inoculated in the morning, by the end of the working day.

B-109 Outcome of a Large Multicenter Prospective Clinical Study Aimed at Validating a Newly Developed VlsE1/pepC10 IgG/IgM Chemiluminescent Immunoassay (CLIA) in Conjunction with a New Automated Analyzer

Abstract Background Lyme borreliosis is the fastest growing vector-borne illness in North America, with recent estimates as high as 476 000 new cases annually in the U.S. alone. Measurement of Borrelia-specific antibodies via serologic testing remains an important diagnostic aid. Since 1994, serologic testing for Lyme disease in the United States has consisted of a two-stage algorithm, termed standard two-tiered testing (STTT). In the STTT algorithm, specimens equivocal/positive by first tier screening methodologies are subsequently tested via western blots. In recent years, the FDA has cleared several alternatives for replacing western blots with more sensitive second-tier tests, termed modified two-tiered testing (MTTT). We recently developed a VlsE1/pepC10 IgG/IgM chemiluminescent immunoassay (CLIA), to be used in conjunction with a new automated analyzer. The goal of these studies was to conduct a multi-site validation for this new assay and analyzer as a first-tier screening test within an STTT algorithm. Methods Retrospective cohort: 280 clinically characterized samples were provided from the U.S. Centers for Disease Control and Prevention (90 cases, 190 controls). The CDC provided VlsE1/pepC10 IgG/IgM ELISA data derived from their own testing. VlsE1/pepC10 IgG/IgM CLIA and western blotting data were also generated for this panel at ZEUS Scientific. Sensitivity and specificity values were calculated for the retrospective cohort as follows:VlsE1/pepC10-CLIA vs existing VlsE1/pepC10 IgG/IgM-ELISA, and VlsE1/pepC10-CLIA + Western blots, vs VlsE1/pepC10 IgG/IgM-ELISA + Western blots, interpreted as STTT algorithms. All testing of the retrospective cohort was performed at ZEUS Scientific. Prospective Cohort: 600 routine Lyme serology specimens were prospectively collected at three locations (1800 total): Mayo Clinic (MOC), Rochester MN.; Marshfield Clinic (MC), Stevens Point, WI; Health Network Laboratories (HNL), Allentown PA. The samples were assayed and analyzed in the same combinations as described above for the retrospective cohort; however, instead of sensitivity and specificity, the results were reported as positive percent agreement (PPA) and negative percent agreement (NPA) since the prospective samples were not clinically characterized. Testing of the prospective cohort was evenly split across three different laboratories, one of which was ZEUS Scientific. Results Retrospective cohort: [VlsE1/pepC10 CLIA, Sensitivity = (77/90) = 85.56%, Specificity = (177/190) = 93.16%; VlsE1/pepC10 ELISA, Sensitivity = (80/90) = 88.89%, Specificity = (179/190) = 94.21%], [STTT-VlsE1/pepC10-CLIA + Western blots, Sensitivity = (72/90) = 80.00%, Specificity = (190/190) = 100.00%; STTT-VlsE1/pepC10-ELISA + Western blots, Sensitivity = (70/90) = 77.78%, Specificity = (190/190) = 100.00%]. Prospective Cohort (1 sample out of the 1800 collected was omitted due to an invalid western blot result):[VlsE1/pepC10-CLIA vs VlsE1/pepC10-ELISA, PPA = (196/235) = 86.51%, NPA = (1452/1564) = 92.84%; STTT-VlsE1/pepC10-ELISA vs STTT-VlsE1/pepC10-ELISA, PPA = (160/167) = 95.81%, NPA = (1607/11 632) = 98.47%]. Conclusion These studies demonstrate that the newly developed VlsE1/pepC10 IgG/IgM CLIA, in conjunction with a new automated analyzer, yielded accurate results as a first-tier screening assay for both the retrospective and prospective multi-site validation cohorts. Future studies, utilizing the same sample cohorts, will be aimed at validating additional second tier CLIA tests as part of an MTTT algorithm.

B-231 Development and Evaluation of a Lyophilized Mpox Virus qPCR Assay

Abstract Background Increased cases of monkeypox virus (MPXV) have been reported to WHO from 110 countries. As of January 16, 2023, a total of 84 733 laboratory confirmed and 1354 probable cases, including 80 deaths, have been reported to WHO. The US is assessed as high-risk region. Real-time PCR, with its sensitivity and specificity, is the preferred lab test technique. Since gene mutation has been observed in MPXV, developing assays with more than one target was recommended by the FDA. Methods A lyophilized qPCR multiplex assay was developed for MPXV detection. This assay contains primers and (1) MGB probe targeting highly conserved MPXV sequence (GeneBank AF380138 F3L), (2) BHQ probe targeting non-variola orthopoxvirus (OPX) sequence, and (3) a probe targeting a selected human house-keeping gene as an internal control. Furthermore, external positive and negative controls were included. A MPXV positive clinical sample from human skin lesions was used for the assay validation. The viral copy number of the sample was determined as 1×107 copies/mL using the standard curve method. Two hundred µL of samples were processed in the KingFisherTM Flex system. The PCR reaction consisted of 5 µL of DNA sample and 20 µL reaction mix and was run on 7500 Fast Dx Real-Time PCR System (Life Technologies). The limit of detection (LoD) was determined as the concentration at which at least 95% of samples were tested positive. A total of 230 contrived samples with concentrations lower than LoD were used to determine cut-off value. Inclusivity and exclusivity studies were conducted by in silico analysis of 80 sequences of MPXV (clade I), 3026 of MPXV (clade II), 10 of camelpox virus, 93 of cowpox virus, 6 of ectromelia virus, and 116 of vaccinia virus (NCBI and GISAID accessed on 10/03/22). Clinical evaluation of the assay was conducted using 80 contrived clinical samples in unique negative skin lesion matrix, including 40 positives (five samples at 5× LOD, five at 2.5×, ten at 1.5×, and twenty at 1×), along with 40 negatives. The positive (PPA) and negative percent agreement (NPA) of the assay were calculated. The study was approved by BRANY IRB. Results The qPCR assay had a cut-off Ct value of 40, and a LoD of 500 copies/mL for both MPXV and OPX with good inclusivity and exclusivity. Clinical evaluation for MPXV yielded a PPA and NPA of 94.9% (95% CI: 83.1%–98.6%) and 100% (89.9%–100%), respectively. For OPX, the PPA and NPA were 92.3% (79.7%–97.4%) and 100% (89.9%–100%), respectively. Conclusions Both analytical and clinical performances of the developed MPXV qPCR assay were good. Lyophilized reagents contain all required components, providing longer shelf-life and easier transportation. MPXV is the only member of the OPX genus known to be circulating among humans in the US currently. This assay as designed could potentially cross-detect different non-variola OPX, such as vaccinia virus and cowpox virus. Healthcare providers should always contact the local public health authorities for more guidance. Furthermore, this study was limited to lesion swab specimens and performance evaluation with additional specimen types is warranted.

A-285 Performance Evaluation of LIAISON® QuantiFERON®-TB Gold Plus Assay and Its Use in Long-Term Care Facilities

Abstract Background Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis with 7860 cases reported in 2021 in the United State and estimated 13 million people are living with Latent TB infection (LTBI); Early detection of TB infection is crucial to prevent the spread of the disease; there are two types of tests for TB and LTBI: Tuberculin Skin Test (TST) and Interferon-Gamma Release Assay (IGRA). Although TST has good sensitivity but false-negative reactions occur in certain population and the specificity of is decreased among persons with prior Bacillus Calmette-Guérin (BCG) vaccination, especially those vaccinated post-infancy and those with repeat vaccination. Similarly, persons living in areas where nontuberculous mycobacteria are common are at increased risk of having false-positive TST reactions. IGRA is blood-based laboratory test that measures responses to TB-specific peptide antigens in whole blood. Like the tuberculin skin test (TST), it is an indirect test for M. tuberculosis infection, but is more specific than TST and is unaffected by prior BCG vaccination and most environmental mycobacterial infections. Methods The LIAISON® QuantiFERON®-TB Gold Plus assay is an in vitro diagnostic test for the detection of interferon-γ (IFN-γ) in human lithium heparin plasma by chemiluminescence immunoassay (CLIA) using the LIAISON ® XL Analyzer. The clinical specificity and sensitivity were 96.9% and 84.4% respectively. Clinical studies have shown Positive Percent Agreement (PPA) of 97.4% and Negative Percent Agreement (NPA) of 98.6%. We evaluated the precision, reproducibility, and correlated patient results to our reference lab results. 452 samples collected from Long-Term Care facilities resident were run over 9 months. Statistical analyses were done using Analyse-it. Results The percentage agreement for precision, reproducibility, and accuracy were 100%. The correlation with the reference laboratory was 100% agreement. We found 14.8% of samples tested were positive, 83.2% were negative and 2.0% were indeterminate. Conclusion The LIAISON® QuantiFERON®-TB Gold Plus assay offers the benefit of a fully automated with high sensitivity and specificity. The faster turnaround will aid the physician in the diagnosing M. tuberculosis, eliminates the need for two-step testing, and is preferred in patients who received BCG vaccination. In addition, with the growing geriatric population, the availability of this test will give the physician the opportunity to follow the 2016 American Thoracic Society (ATS)/Infectious Disease Society of America (IDSA)/CDC clinical practice guidelines for using IGRAs in older patients.

B-011 Analytical Validation of a New Vasoactive Intestinal Peptide Radioimmunoassay

Abstract Background Vasoactive intestinal peptide (VIP), a potent vasodilator, is produced by neuronal cells located in the central nervous system, the digestive, respiratory, and urogenital tracts, and the exocrine, thyroid, and adrenal glands. Increased circulating VIP is clinically useful to detect VIP-producing tumors in patients with chronic diarrheal diseases. Objective Develop a sensitive replacement radioimmunoassay (RIA) for VIP quantitation in human plasma due to the diminished supply of the antibody used in the previous in-house method. Method Patient VIP in EDTA plasma competes with labeled (125I) VIP for a limited number of primary rabbit polyclonal antibody (BMA Biomedicals, Augst, Switzerland) binding sites. Antibody-bound VIP is then complexed with a goat anti-rabbit secondary antibody and precipitated out of the solution with the use of polyethylene glycol and centrifugation. After the supernatant is discarded, the 125I signal from the pellet is counted on the Wizard2 gamma counter (Perkin Elmer, Waltham, MA). The measured signal is inversely proportional to the amount of VIP present in the patient’s sample. Method validation of the new assay included determination of imprecision (pooled human EDTA plasma), limits of detection and quantification, analytical measurement range (AMR), accuracy by spike recovery, interferences, cross-reactivity, stability, reference interval determination, and agreement with the current Mayo laboratory-developed assay. Results Stability studies on freshly collected EDTA plasma showed VIP is stable for 3 days ambient, 7 days refrigerated, and 14 days frozen. Intra-assay imprecision studies showed 8.3, 2.3, and 1.3%CV for mean VIP concentrations of 41, 144, and 304 pg/mL. Inter-assay imprecision was 12.9, 7.6, 5.2, and 7.5%CV for mean VIP concentrations of 30, 81, 180, and 308 pg/mL. The assay limit of detection was 29 pg/mL. The limit of quantitation was 30 pg/mL (%CV = 13%) and was established via precision profile. The AMR was 30–500 pg/mL (slope of 0.98, intercept of 3.0 pg/mL, and R2 of 0.97). The mean % spike recovery using a different preparation lot of calibrator material was 89% (range 83%–93%). The assay was not affected by up to 1000 mg/dL hemoglobin, 1000 mg/dL triglycerides, or 10 mg/dL bilirubin. Peptide histidine methionine 27, neurotensin, gastric inhibitory peptide, motilin, secretin, and glucagon do not cross-react in the assay. The 97.5th reference limit was <86 pg/mL (95% CI 81–90, n = 127). Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) with the current Mayo VIP assay were 97, 82, and 90% respectively. Conclusion We have developed a replacement VIP RIA. This assay has a lower limit of quantitation and a broader AMR than the current assay. Concordance with the current assay was acceptable.

A-267 Evaluation of the Quidel Sofia 2 Rapid Lyme Test in a Modified Two-tier Testing Protocol

Abstract Background Several Modified two-tier testing algorithms (MTTT) for Lyme disease involving sequential enzyme immunoassays (EIA) have been approved by the FDA as an alternative to conventional two-tiered testing using EIA followed by immunoblot. The MTTT algorithms are reported to be more sensitive than conventional two-tiered algorithm’s especially in early Lyme disease. The Quidel Sofia 2 Lyme assay utilizes lateral flow with fluorescence detection (FIA) providing a rapid on-demand instrument-read alternative to central laboratory batch mode testing We compared the Quidel assay as a first-tier test in an MTTT algorithm to the Zeus Scientific VlsE1/pepC10 IgG/IgM enzyme immunoassay (EIA) followed by the Zeus monovalent IgM and IgG EIA as the confirmatory test. Methods Serum specimens (n = 179) were obtained from consecutive samples sent to our clinical laboratory for Lyme serology testing. For the first-tier the samples were tested using the Zeus EIA VlsE1/pepC10 lgG/IgM test and the Quidel Sofia 2 Lyme FIA assay. Positive or equivocal first-tier tests were then tested using the Zeus monovalent IgM and IgG EIA as the confirmatory test. Results The positive percent agreement was 100%, the negative percent agreement 97.9 % with an overall agreement of 98.3%. The Kappa statistic was 0.945 (95% confidence interval 0.883–1.000 indicating “almost perfect” agreement). Conclusion The Quidel Sofia Lyme test performs well when compared to the Zeus test in a two-tiered testing MTTT algorithm. Obtaining samples to evaluate Lyme serologic tests can be challenging. Our data should prove useful for facilities considering the use of the Quidel assay.

A-252 Differentiation of SARS-CoV-2, Influenza A and B, and Respiratory Syncytial Virus Using a Single Swab: A Clinical Performance Evaluation of the BD MAX System Multiplex PCR Assay

Abstract Background Some shared clinical features of SARS-CoV-2, influenza (flu), and respiratory syncytial virus (RSV) infection often pose a diagnostic challenge for clinicians. In that context, the use of molecular diagnostic systems multiplex assays, using a nasopharyngeal or nasal swab, facilitates the detection of the causing virus by differentiating between the RNA of each disease. Our study evaluated the clinical accuracy of one such multiplex PCR assay, the BD Respiratory Viral Panel for BD MAX™ System (BD MAX RVP). Methods Symptomatic patients that had a nasopharyngeal and nasal specimen prospectively collected from 6 geographical locations in the US were enrolled in the study between January and April 2022 along with nasopharyngeal swabs retrospectively collected between December 2019 and January 2022. All specimen testing was performed using the BD MAX System. Hologic® Flu A/B/RSV assay on Panther Fusion™ System was the comparator for flu A, flu B, and RSV while a composite of two out of three assays authorized under FDA Emergency Use Authorization (Roche cobas® SARS-CoV-2 on cobas® 6800 System, Hologic® Aptima® SARS-CoV-2 on Panther® System, and Quidel Lyra® SARS-CoV-2 on Applied Biosystems® 7500 Fast Dx) was used as comparator for SARS-CoV-2. Positive and negative percentage agreements (PPA and NPA, respectively) were calculated to determine performance. Reproducibility and precision assessments were also performed as part of the study. Results Tested specimens included 252 nasopharyngeal and 254 nasal swabs, prospectively collected, and 240 retrospectively acquired nasopharyngeal specimens. For prospectively collected SARS-CoV-2 nasopharyngeal specimens, PPA of 98.8% (95% CI 93.6, 99.8) and NPA of 98.2% (95% CI 94.9, 99.4) were observed while for flu A, PPA was 100% (95% CI 61.0, 100) and NPA 99.6% (95% CI 97.7, 99.9). PPA and NPA for prospectively collected SARS-CoV-2 anterior nasal specimens were 98.8% (95% CI 93.3, 99.8) and 98.3 (95% CI 95.1, 99.4), respectively. For flu A, PPA of 100% (95% CI 61.0, 100) and NPA of 99.6% (95% CI 97.8, 99.9) were observed. Lack of enrolled positive patients with flu B or RSV precluded the PPA calculation for those viruses. However, NPA for both flu B and RSV was 100% for the two specimen types. In retrospectively obtained nasopharyngeal specimens, PPA was 100% (95% CI 93.7, 100) for flu A, 100% (95% CI 93.8, 100) for flu B, and 98.4% (95% CI 91.5, 99.7) for RSV while NPA was 98.9% (95% CI 96.1, 99.7) for flu A, 98.9% (95% CI 96.1, 99.7) for flu B, and 100% (95% CI 97.9, 100) for RSV. Conclusion The BD MAX RVP assay showed a strong clinical performance in detecting and differentiating between the RNA of SARS-CoV-2, flu A and B, and RSV.

B-056 Performance Characteristics of a New, Fully Automated Planar Microarray Immunoassay for the Detection of IgG Auto-Antibodies Against Centromere Protein B (CENP-B)

Abstract Background Detection of auto-antibodies against Extractable Nuclear Antigens (ENA), including CENP-B, is key in identification of autoimmune (AI) diseases. Many current test methods are manual, slow, labor intensive or do not facilitate the simultaneous detection of several auto-antibodies, that could reduce time to provide comprehensive results to clinicians. We report the performance characteristics of MosaiQTM CENP-B (Quotient Suisse, Eysins, CH), a new planar microarray immunoassay designed for use with the fully automated, continuous random access, high throughput MosaiQ system for qualitative serological detection of CENP-B IgG auto-antibodies. CENP-B assay is one parameter of a multiplexed MosaiQ connective tissue disease panel in development. Methods MosaiQ CENP-B microarrays were prepared by printing CENP-B antigens onto epoxy-silane glass chips and assembled into magazines for processing on the MosaiQ 125 instrument (Fig. 1). Primary patient serum tubes and reagents are loaded on the instrument and automatically processed to generate immunoassay spot signals, interpreted by the instrument using a proprietary image analysis algorithm. Anonymized human serum samples (Hôpital Pitié Salpetrière, Paris, France), characterized as CENP-B auto-antibodies non-reactive or reactive with ≥1 CE-marked devices, were included in the study. Results After discordant analysis, MosaiQ system correctly identified 96/99 samples characterized as reactive by comparators and all 199 non-reactive samples: positive percent agreement 97.0% (95% CI, 91.4%, 99.4%), negative percent agreement 100% (95% CI, 98.2%, 100%), overall percent agreement 99% (95% CI, 97.1%, 99.8%). A reproducibility and repeatability evaluation showed, respectively, overall agreement of 99.7% and 100%. Conclusion MosaiQ CENP-B microarray provides a high level of clinical concordance with other CE-marked assays for detecting CENP-B auto-antibodies. The MosaiQ System has the potential to advance AI disease testing by increasing laboratory efficiency and productivity, with the ability to multiplex the detection of various auto-antibodies on a single microarray, and a capacity to automatically process thousands of samples per day.

B-104 Comparison of the Performance Characteristics of the Third Generation Anti-Cyclic Citrullinated Peptide CCP3.1 IgG/IgA and CCP3 IgG Assays

Abstract Background Rheumatoid arthritis (RA) is one of the most common systemic autoimmune diseases. Anti-citrullinated protein antibody (ACPA) is the most specific serological biomarker for RA and is associated with significantly more disease activity compared to rheumatoid factor (RF). Improved autoantibody detection relied in the generation of tests against cyclic citrullinated peptide (CCP), a subset of ACPA. The 3rd generation anti-CCP IgG antibody (CCP3) emerged with increased sensitivity compared to the earlier versions. More recently, CCP3.1 with conjugate to detect IgA antibodies in addition to the usual IgG antibodies became available. This is described to exhibit enhanced sensitivity due to early RA patients producing IgA antibodies to CCP3 in the absence of IgG. In this study, we evaluated the diagnostic characteristics of CCP3.1 and its association with disease severity in comparison to CCP3. Methods Our cohort included a total of 331 subjects submitted for RA panel or independent antibody testing. Chart review revealed 136 clinically defined RA- positive and 195 RA- negative patients. Residual sera from these patients were tested for RF (Roche Diagnostics, Indianapolis, US), CCP3 and CCP3.1 (Inova Diagnostics, San Diego, US) antibodies. Clinical performance characteristics at the manufacture-suggested cut-off and association with disease severity were assessed and compared between the two assays. Results The overall diagnostic accuracy was comparable between the two assays with CCP3 exhibiting an area under the curve (AUC) of 0.879, 95% CI: 0.836–0.922, and CCP3.1 showed an AUC of 0.890, 95% CI: 0.849–0.931. CCP3 displayed a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 78.7%, 90.8%, 85.6% and 85.9% respectively. CCP 3.1 displayed an equivalent performance with 77.9% sensitivity, 93.3% specificity, 89.1% PPV and 85.8% NPV. Both assays showed an excellent agreement with a positive percent agreement of 92.0% and negative percent agreement of 98.7%. There was a strong quantitative correlation between the two assays (Spearman correlation coefficient, r = 0.864, P < 0.001). Paired t test showed significantly higher titers of CCP3 (mean:118, median:145) compared to CCP3.1 (mean:110, median: 108) in RA patients. RF and CCP3.1 combination yielded a slightly greater specificity (99.0%), positive likelihood ratio (65.9) and odds ratio (201.7) compared to the RF and CCP3 combination (specificity: 98.5%, positive likelihood ratio: 44.5, odds ratio: 138.4). Nine early-RA patients (with symptom onset <6 months of diagnosis) were detected by both assays and displayed comparable titers (CCP3.1, mean:138, median:149; CCP3, mean:149, median:161). Out of 60 RA patients exhibiting more severe disease course with the presence of bone erosions, 50 of them were detected by CCP3.1 (mean:125, median:146) and 51 were detected by CCP3 (mean:139, median:151) and the titers were comparable. Conclusion CCP3 and CCP3.1 assays displayed comparable performance for detection of RA and correlated similarly with the disease severity. In our study cohort that mainly comprised of RA patients diagnosed >6 months after symptoms onset, addition of IgA isotype showed no added value but did not compromise performance.

A-372 Increased Sensitivity in Detection of SARS-CoV-2 Antigen Using RADI COVID-19 Ag Rapid Test

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) rapid point-of-care antigen tests can provide easy and fast results for the diagnosis of coronavirus disease 2019 (COVID-19). However, due to the lower sensitivity of rapid antigen tests relative to reverse transcription polymerase chain reaction (RT-PCR), they might miss many infectious COVID-19 cases. We evaluated and compared a new rapid antigen test with the current approved one. Methods The RADI COVID-19 Ag Rapid Test (KH Medical, Hanam, Korea) was validated with a total of 1054 pre-confirmed residual nasopharyngeal swab samples (258 positives and 796 negatives) by PowerChek 2019-nCoV Real-time PCR (Kogene Biotech, Seoul, Korea). Among them, 258 positives and 546 negatives were used to compare the RADI COVID-19 Ag Rapid Test with the STANDARD Q COVID-19 Ag Test (SD Biosensor, Suwon, Korea). Results Using RT-PCR results as the reference, the overall sensitivity and specificity of RADI COVID-19 Ag Rapid Test were 82.2% (212/258; 95% confidence interval, CI [76.9–86.6]) and 99.1% (789/796; 95% CI [98.2–99.7]), respectively. The positive predictive value and negative predictive value were 96.8% (212/219; 95% CI [93.5–98.5]) and 94.5% (789/835; 95% CI [93.0–95.7]), respectively. The accuracy was 95.0% (1001/1054; 95% CI [93.5–96.2]) with Cohen's kappa value of 0.86 (95% CI [0.82–0.89]). The positive percent agreement and negative percent agreement of the RADI COVID-19 Ag Rapid Test for STANDARD Q COVID-19 Ag Test were 97.0% (159/164; 95% CI [93.0–99.0]) and 90.6% (580/640; 95% CI [88.1–92.8]), respectively, and those of STANDARD™ Q COVID-19 Ag Test for RADI COVID-19 Ag Rapid Test were 72.6% (159/219; 95% CI [66.2–78.4] and 99.2% (580/585; 95% CI [98.0–99.7], respectively. The overall percent agreement between the two rapid antigen assays was 91.9% (739/804; 95% CI [89.8–93.7]) with Cohen's kappa value of 0.78 (95% CI [0.73–0.83]). Among the 65 discrepant samples between the two rapid antigen assays, 56 were positive as per RT-PCR, which showed 53 samples negative for STANDARD Q COVID-19 Ag Test and 3 samples negative for RADI COVID-19 Ag Rapid Test. The median E gene Ct value of the 65 discrepant samples was 28.2 (interquartile range: 26.1–32.3). Conclusion RADI COVID-19 Ag Rapid Test provides acceptable sensitivity and specificity according to the validation criteria for KFDA immunological test product approval. Furthermore, RADI COVID-19 Ag Rapid Test shows improved sensitivity compared to the current approved rapid antigen test.

Extraction-Free Detection of SARS-CoV-2 Viral RNA Using LumiraDx's RNA Star Complete Assay from Clinical Nasal Swabs Stored in a Novel Collection and Transport Medium.

Background: The rapid detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is vital for patient care. The LumiraDx™ SARS-CoV-2 RNA Star Complete (RSC) is an Emergency Use Authorization-recognized molecular test using nasal/nasopharyngeal swabs immersed in a viral/universal transport medium (VTM/UTM). However, there is a critical need for an alternative medium for point-of-care testing (POCT). This study aimed to investigate Xtract-Free (XF), a novel collection medium for transport and direct (extraction-free) use with nucleic acid tests. Methods: Using serially diluted SARS-CoV-2 viral RNA (vRNA) in a routine UTM and XF, a limit of detection (LOD) was established via an RSC test and a quantitative reverse transcription PCR (RT-qPCR). Additionally, the results obtained from a panel of 108 clinical "car-side" nasal swabs collected in XF during the coronavirus pandemic and assessed using the "gold-standard" RT-qPCR assay were compared to Lumira's RSC assay. Results: The average replicate RT-qPCR cycle threshold (CT) values for vRNA in XF and UTM were observed to be equivalent. An LOD for which five out of five replicates were detected using XF or VTM was approximately 2000 copies/mL. The nasal swabs collected in XF exhibited 93.9% positive percent agreement (sensitivity) and 100% negative percent agreement (specificity) compared to the RT-qPCR. Three specimens tested positive via an RT-qPCR were negative when tested via RSC; however, all three samples had CT values ≥ 36.4. Conclusions: XF is equivalent to VTM/UTM and is compatible for use with the RSC test. Furthermore, XF can be used directly with RT-qPCRs and rapid antigen testing without the requirement for separate nucleic acid extraction (an extraction-free process), making it ideal for cost-effective high-throughput and decentralized respiratory testing. Impact Statement: This study is the first to evaluate LumiraDx's SARS-CoV-2 RNA Star Complete assay in concert with Xtract-Free (XF), a novel collection medium containing a proprietary RNase-inactivating technology for the rapid, "extraction-free" detection of SARS-CoV-2 RNA from clinical nasal swabs. Specimens collected in XF combined with rapid LumiraDx detection provide a safe and sensitive alternative to VTM/UTM, and Molecular Transport medium (MTM) for high throughput, "extraction-free" molecular detection.

Evaluation of the QIAstat-Dx Meningitis/Encephalitis Panel, a multiplex PCR platform for the detection of community-acquired meningoencephalitis.

Rapid identification of the causative pathogens of central nervous system infections is essential for providing appropriate management and improving patient outcomes. The performance of QIAstat-Dx Meningitis/Encephalitis (ME) Panel-a multiplex PCR testing platform-in detecting pathogens implicated in meningitis and/or encephalitis was evaluated using BioFire FilmArray ME Panel as a comparator method. This multicenter study analyzed 585 retrospective residual cerebrospinal fluid specimens and 367 contrived specimens. The QIAstat-Dx ME Panel showed positive percent agreement (PPA) values of 100% for Neisseria meningitidis, Streptococcus agalactiae, Escherichia coli K1, Listeria monocytogenes, and Cryptococcus gattii/neoformans on clinical samples compared to the BioFire FilmArray ME Panel. The PPA values observed for Haemophilus influenzae and Streptococcus pneumoniae were 80% and 88.24%, respectively. Negative percent agreement (NPA) values were >99.0% for each of the six bacterial targets and one fungal target tested with clinical samples. One viral target, herpes simplex virus 1, exhibited a PPA value of 100.0%, while the remaining viral targets-human parechovirus, herpes simplex virus 2, human herpes virus 6, and varicella zoster virus-were >90.0%, with the exception of enterovirus, which had a PPA value of 77.8%. The QIAstat-Dx ME Panel detected five true-positive and four true-negative cases compared to BioFire FilmArray ME Panel. The NPA values for all viral pathogens were >99.0%. Overall, the QIAstat-Dx ME Panel showed comparable performance to the BioFire FilmArray ME Panel as a rapid diagnostic tool for community-acquired meningitis and encephalitis.

Development of a Rapid and High-Throughput Multiplex Real-Time PCR Assay for Mycoplasma hominis and Ureaplasma Species

Bacterial commensals of the human genitourinary tract, Mycoplasma hominis and Ureaplasma species (parvum and urealyticum) can be sexually transmitted, and may cause nongonococcal urethritis, pelvic inflammatory disease, and infertility. Mycoplasma hominis and Ureaplasma species may also cause severe invasive infections in immunocompromised patients. Current culture-based methods for Mycoplasma/Ureaplasma identification are costly and laborious, with a turnaround time between 1 and 2 weeks. We developed a high-throughput, real-time multiplex PCR assay for the rapid detection of M. hominis and Ureaplasma species in urine, genital swab, body fluid, and tissue. In total, 282 specimens were tested by PCR and compared with historic culture results; a molecular reference method was used to moderate discrepancies. Overall result agreement was 99% for M. hominis (97% positive percentage agreement and 100% negative percentage agreement) and 96% for Ureaplasma species (96% positive percentage agreement and 97% negative percentage agreement). Specimen stability was validated for up to 7 days at room temperature. This multiplex molecular assay was designed for implementation in a high-complexity clinical microbiology laboratory. With this method, >90 samples can be tested in one run, with a turnaround time of 4 to 5 hours from specimen extraction to reporting of results. This PCR test is also more labor effective and cheaper than the conventional culture-based test, thus improving laboratory efficiency and alleviating labor shortages.

Evaluation of the fully automated LIAISON®XL chemiluminescence analyzer for QuantiFERON®-CMV testing in transplant recipients.

Monitoring CMV-specific cell-mediated immunity by the QuantiFERON®-CMV (QF-CMV) may be useful in predicting the risk of CMV infection in transplant recipients (TR). As the QuantiFERON-Tuberculosis (QFT®-Plus) became available on the fully automated LIAISON®XL chemiluminescence (CLIA) analyzer, we evaluated the performance of the QF-CMV on the LIAISON®XL analyzer using the QuantiFERON®-TB Gold Plus reagent. Between 2018 and 2022, 81 samples from TR were collected at the Department of Virology of Limoges Hospital, France. Whole blood was collected into each of the three QF-CMV collection tubes: a CMV-antigen tube (QF-Ag), a mitogen tube (QF-Mg) (positive control), and a nil tube (negative control). The QF-CMV was performed on the LIAISON®XL analyzer, and results were compared with those obtained by conventional microplate ELISA. Intra- and inter-assay coefficients of variation were inferior to 20%. No inter-sample contamination was found (p=0.366). The level of concordance between CLIA and the commonly used ELISA method was 88.9%. Positive and negative percent agreements were 92.3% and 85.7%, respectively, with a very good agreement between assays (κ=0.818). Most discordances were due to indeterminate- or negative-ELISA/positive-CLIA results (most of ELISA results were borderline). Linear regression analyses demonstrated a strong correlation between both assays (QF-Ag Pearson's r=0.978, QF-Mg Pearson's r=0.963). No significant difference was observed in median QF-CMV values between both assays (QF-Ag p=0.776; QF-Mg p=0.853; Mann-Whitney U test). The Bland-Altman plots showed a minor difference in IFN-γ release (QF-Ag -0.069 IU/ml, 95% limits of agreement (LoA): -1.589; 1.451; QF-Mg0.190IU/ml, 95% LoA: -2.070; 2.450). Automated QF-CMV with CLIA is comparable to QF-CMV performed by ELISA with a presumably higher sensitivity for IFN-γ detection that may result in the conversion of samples close to the ELISA cut-off into positive results. Moreover, the use of a random-access analyzer allows to optimize the follow-up of TR.

Development of a rapid lateral flow assay for detection of anti-coccidioidal antibodies.

Coccidioides spp. are dimorphic fungi that are capable of infecting human and non-human mammals and can cause diverse manifestations of coccidioidomycosis or Valley fever (VF). In combination with clinical symptoms and radiographic findings, antibody-based diagnostic tests are often used to diagnose and monitor patients with VF. Chitinase 1 (CTS1) has previously been identified as the seroreactive antigen used in these diagnostic assays to detect anticoccidial IgG. Here, an indirect enzyme-linked immunosorbent assay to detect IgG to CTS1 demonstrated 165 of 178 (92.7%) patients with a positive result by immunodiffusion (ID) and/or complement fixation (CF) had antibodies to the single antigen CTS1. We then developed a rapid antibody lateral flow assay (LFA) to detect anti-CTS1 antibodies. Out of 143 samples tested, the LFA showed 92.9% positive percent agreement [95% confidence interval (CI), 84.3%-96.9%] and 97.7% negative percent agreement (95% CI, 87.9%-99.6%) with ID and CF assays. Serum or plasma from canines, macaques, and dolphins was also tested by the CTS1 LFA. Test line densities of the CTS1 LFA correlated in a linear manner with the reported CF and ID titers for human and non-human samples, respectively. This 10-min point-of-care test for the rapid detection of anti-coccidioidal antibodies could help to inform healthcare providers in real-time, potentially improving the efficiency of healthcare delivery.

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.

Diagnostic performance of respirators for collection and detection of SARS-CoV-2

Respirators, called as face mask, have been used to protect the wearer from the outside harmful air environment and prevent any virus from being released to neighbors from potentially infected exhaled breath. The antiviral effectiveness of respirators has not only been researched scientifically, but has also become a global issue due to society's obligation to wear respirators. In this paper, we report the results of a study on the collection and detection of viruses contained in exhaled breath using respirators. The inner electrostatic filter was carefully selected for virus collection because it does not come in direct contact with either human skin or the external environment. In the study of a healthy control group, it was confirmed that a large amount of DNA and biomolecules such as exosomes were collected from the respirator exposed to exhalation, and the amount of collection increased in proportion to the wearing time. We conducted experiments using a total of 72 paired samples with nasopharyngeal swabs and respirator samples. Out of these samples, fifty tested positive for SARS-CoV-2 and twenty-two tested negative. The PCR results of the NPS and respirator samples showed a high level of agreement, with a positive percent agreement of ≥ 90% and a negative percent agreement of ≥ 99%. Furthermore, there was a notable level of concordance between RCA-flow tests and PCR when examining the respirator samples. These results suggest that this is a non-invasive, quick and easy method of collecting samples from subjects using a respirator, which can significantly reduce the hassle of waiting at airports or public places and concerns about cross-contamination. Furthermore, we expect miniaturized technologies to integrate PCR detection into respirators in the near future.

Performance comparison of three commercial tests for the detection of SARS-CoV-2 antibodies in a common set of pediatric samples

BackgroundSerologic testing for SARS CoV-2 is useful for detection of past infection and assisting in diagnosis of post-COVID-19 syndromes such as MIS-C. Immune responses to SARS-CoV-2 infection in children differ from adults but most antibody performance studies are limited to adults. ObjectiveThe objective of this study was to compare three commercial SARS-CoV-2 antibody kits in a common set of children being evaluated for SARS-CoV-2 infection. MethodsThree SARS-CoV-2 antibody tests: Abbott anti-nucleocapsid (N) IgG (AA), Epitope Diagnostics anti-N IgG (EDI) and EUROIMMUN anti-S1 Spike IgG (EU) were compared against two references: 1) RT-PCR and 2) consensus IgG (consIgG). ResultsAll three tests had a sensitivity <53% compared to RT-PCR, with EU outperforming EDI (p = 0.03). When all samples were compared to consIgG, positive percent agreement was comparable (AA-90%, EU- 98% and EDI- 88%) but EDI had significantly better negative percent agreement than EU (p = 0.009). No difference in test performance was observed using either reference when samples were collected ≥15 days post-symptom onset (PSO). ConclusionsOur findings suggest good performance of commercial SARS-CoV-2 IgG assays in pediatric patients with samples collected ≥15 days PSO. Additional studies investigating antibody response and assay performance in children are warranted.