Laboratory-developed Tests Research Articles

Performance Assessment of a Multiplex Real-Time PCR Assay for Detection of Viruses Causing Respiratory Tract Infections.

Following the COVID-19 pandemic, global epidemiological trends demonstrate a return to pre-pandemic levels of respiratory syncytial virus (RSV) and influenza (Flu) A/B viruses. For the appropriate clinical management of viral infections, reliable and timely diagnosis is crucial. The clinical presentation of these respiratory viral infections shows significant overlaps; thus, the syndromic diagnosis of these infections becomes challenging. The goal of this study was to compare the performance of three multiplex real-time PCR-based platforms for the detection of SARS-CoV-2, Flu A, Flu B, and RSV. A retrospective study was performed on 200 de-identified nasopharyngeal and oropharyngeal specimens. All samples were tested simultaneously on three PCR-based platforms for the detection of SARS-CoV-2, Flu A, Flu B, and RSV: HealthTrackRx's real-time PCR Open Array® respiratory panel, TrueMark™ SARS-CoV-2, Flu A, Flu B, RSV Select Panel, and BioFire® RP2.1 Panel. The positive and negative predictive value of each test was evaluated at a 95% confidence interval. Among the 200 tested samples, the TrueMark™ and OpenArray® laboratory-developed tests (LDTs) showed a 100% concordance for the detection of SARS-CoV-2, Flu A, Flu B, and RSV. Overall agreement of 100% was observed for nasopharyngeal samples between the laboratory-developed tests and FDA-approved BioFire® RP2.1 Panel. Diagnostic results for these four respiratory viruses, in clinical samples, between the LDTs and the FDA-approved comparator demonstrated full concordance. Respiratory viral infections represent one of the major global healthcare burdens. Consequently, the accurate detection and surveillance of these viruses are critical, particularly when these viruses are known to co-circulate. The excellent performance and full concordance of the LDTs, with the BioFire® Respiratory RP2.1 panel, in detecting SARS-CoV-2, Flu A, Flu B, and RSV shows that these tests can be confidently implemented for the clinical testing of respiratory viral infections.

The US FDA’s New Rule for Regulating Laboratory-Developed Tests

This Viewpoint discusses potential benefits and obstacles regarding US Food and Drug Administration (FDA) oversight of laboratory-developed tests.

6848 Improvement Of Free Thyroxine Measurement Quality Through The CDC Clinical Standardization Programs

Abstract Disclosure: A. Ribera: None. N. Vazquez: None. S. Knoblock: None. O. Sugahara: None. T. Buchannan: None. L. Collins: None. D. Spector: None. L. Zhang: None. U. Danilenko: None. H.W. Vesper: None. Thyroid function tests are among the most requested tests in patient care, and reliability of these measurements are crucial for appropriate diagnosis and management of thyroid disorders. In response to concerns from the clinical and research community about the quality of thyroid function tests, the CDC Clinical Standardization Programs (CSP) has created the hormone standardization (HoSt) program for free thyroxine (FT4) in partnership with the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC). As a first step, CDC CSP has completed an interlaboratory comparison study of commercial immunoassays (IA) and lab-developed tests (LDT) to determine the current agreement among assays. Participants received 40 blinded, high quality individual donor serum samples with FT4 reference values assigned by the CDC FT4 reference measurement procedure (RMP). FT4 concentrations ranged between 11-32 pmol/L. Samples were analyzed in duplicate over 2 days and results were compared to the CDC FT4 RMP. To assess the potential impact of standardizing measurements to the CDC HoSt FT4 program, reported results were normalized to the CDC RMP results (recalibrated in silico using linear regression). Mean percent bias to the reference value was evaluated pre- and post-recalibration. In addition, inter-assay agreement of classification (assignment of hypo-, eu- or hyperthyroid status according to FT4 reference intervals) were assessed pre- and post-recalibration using the assay-specific and CDC RMP reference intervals, respectively. A total of 19 methods participated, consisting of 15 IA and 4 LDT. All participants showed a negative mean bias to the RMP, with IA showing a higher magnitude of bias (average: -20.7 ± 0.9%) than LDT (average: -4.7 ± 1.1%). Of the 40 samples measured, only 53% were classified uniformly by all 19 participants. Post-recalibration, mean percent bias improved profoundly (IA average: 0.0 ± 0.8%, LDT average: 0.1 ± 1.1%), and classification agreement improved by 33% to 86% of samples with consistent classification. The CDC CSP interlaboratory comparison among FT4 assays highlights the critical need for measurement standardization to improve accuracy of FT4 measurements and increase consistency in result interpretation. This study demonstrates that it is possible to develop common FT4 reference intervals via measurement standardization, which would allow for universal evidence-based clinical guidelines to be used in patient care. Presentation: 6/3/2024

B-189 Alinity m You-Create Lab-Developed Test for hepatitis delta virus

Abstract Background Lab developed tests (LDTs) play an important role in the realm of infectious diseases. Current molecular diagnostic platforms offer fully automated high-throughput performance capable of running LDTs. The Alinity m platform enables users to run LDTs concurrently with other FDA cleared or approved IVD assays with its Alinity m You-Create feature. Here, we investigated the performance of an LDT assay targeting hepatitis delta virus (HDV) using commercially available PCR reagents. Methods Alinity m You-Create LDT for HDV was evaluated using Applied Biosystems TaqMan Fast Virus 1-Step Multiplex Master Mix, No ROX, (ThermoFisher, Cat: 5555532). Sample extraction utilized Alinity m Sample Prep Kit 2. Alinity m You-Create HDV LDT analytical performance for sensitivity, linearity and precision was assessed using panels prepared by diluting the HDV 1st WHO international standard in negative plasma. Alinity m You-Create HDV LDT clinical performance was assessed by comparing 45 HDV positive samples previously tested on the m2000 HDV LDT. Assay specificity was assessed testing 97 HDV negative, HBV and/or HCV positive (by serology or nucleic acid amplification test). Results Alinity m You-Create HDV LDT had 100% detection as low as 2.5 IU/mL with SD ≤0.25 Log IU/mL for panels that were ≥2.5 IU/mL. Coefficient of correlation between Alinity m You-Create HDV LDT and m2000 HDV LDT was 0.969 and the mean bias was -0.16 Log IU/mL. All HDV negative specimens were undetected by Alinity m You-Create HDV LDT. Conclusions This study demonstrated successful implementation of an LDT for HDV on the Alinity m platform using the Alinity m You-Create feature using commercially available PCR reagents. The Alinity m You-Create feature for HDV has not been approved by FDA for use in the detection or diagnosis for HDV and its safety and effectiveness has not been established.

A-075 Comparability of Free Testosterone Measurements- Results of the Interlaboratory Study

Abstract Background Assessment of free testosterone (FT) has been recommended as a biomarker for diagnosis and management of hypogonadism and female hyperandrogenism. Reliable laboratory measurements are essential for correctly diagnosing and treating these diseases. FT is currently measured using direct commercial immunoassays, assays employing equilibrium dialysis (ED) or other separation techniques, or it is estimated by calculating free testosterone (cFT). There are concerns about the accuracy and reliability of current methods that may impact patient care. To obtain more information about variability of FT measurements used in patient care, CDC’s Clinical Standardization Programs (CDC CSP) conducted an interlaboratory comparison study. Methods CDC CSP developed an accurate, automated method using ED coupled with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ED-UHPLC-MS/MS). The ED step follows an internationally recognized procedure. The method is calibrated with primary reference material (National Measurement Institute-M914) and was used to assign target values to sera used in the study. The FT concentrations ranged from 3 to 339 pg/mL. In the interlaboratory comparison study single-donor serum samples were provided to participants operating FT methods used in patient care. The participating methods included commercially available platforms utilizing Chemiluminescence Immunoassay or radioimmunoassay techniques, as well as laboratory-developed assays using ED-LC-MS/MS. Results Preliminary findings of the CSP interlaboratory comparison study found large variability among participating assays. The agreement among IA and among ED-LC-MS/MS assays was close. However, FT concentrations obtained by IAs were in average 6 times lower compared to the ED-LC-MS/MS methods. Results estimated using the Vermeulen equation overestimated FT in average by 15% compared to the ED-LC-MS/MS methods and by over 6 times compared to IAs. Conclusions Large variability is observed between FT laboratory developed tests (LDTs) and IAs. As both LDTs and IAs are used in patient care, standardization of FT measurements to assure better patient care is needed. CDC CSP is addressing the need by developing highly accurate and precise FT method and conducting an interlaboratory comparison studies that can aid with improvement of the agreement among assays and identifying potential factors causing differences among assays.

B-025 Revolutionizing NGS-driven Laboratory Developed Tests: The impact of automated liquid handling on plate-based bead clean-up

Abstract Background Automated liquid handling systems are proving to be a transformative technology in the landscape of Laboratory Developed Tests (LDTs), especially those utilizing Next-Generation Sequencing (NGS). By enhancing accuracy, boosting efficiency, and offering scalable solutions, these systems enable labs to meet current and future challenges more effectively. The right system not only improves the quality of diagnostics but also positions labs to be agile and adaptive in a rapidly advancing healthcare environment. firefly® is a well-suited liquid handling solution for diagnostics labs developing LDTs due to its compact footprint, user-friendly software and exceptional versatility. Validated protocol templates that support NGS library preparation are readily available from the firefly cloud and can be adapted to meet specific lab needs. For example, automated bead-based clean-ups designed for 96- and 384- NGS applications are available on firefly. These remove DNA fragments of undesirable sizes, enzymes or primers rom the amplified DNA after a PCR reaction. Automating this process facilitates higher throughput and scaling. firefly has a unique combination of 6 positive displacement dispense heads and a 96/384-well air-displacement pipetting head, making it ideal to distribute the reagents required for bead clean-ups. Methods firefly protocols were developed and optimized to be compatible with a range of elution volumes and bead ratios. Protocol performance was tested using library DNA (∼300bp). The same input DNA was plated out and quantified using the QuantiFluor dsDNA system (Promega) before and after running the protocol. This data was used to assess any reduction in DNA concentration and uniformity across the plate. The protocol's ability to successfully modulate the fragment size distribution was demonstrated using a 50bp DNA ladder and different bead ratios. Data was obtained using a Fragment Analyzer (Agilent, DNF-474 HS NGS Fragment Kit). Well-to-well contamination was assessed by running no-template-controls (NTCs) in wells adjacent to library DNA, then analyzed by qPCR using a LightCycler 480 System (Roche, KAPA Library Quantification kit). Results 96 format has a setup time of 10 minutes, run time of 43 and %CV library concentration of <6%. 384 format has a setup time of 10 minutes, run time of 47 and %CV library concentration of <8%. Uniformity of concentration and fragment sizes was seen across the plates. Comparison of the library concentration before and after the clean-up showed a 5% reduction in DNA concentration after the 96-well plate clean-up and an 11% reduction in library concentration when using the 384-well plate clean-up. Fragment size analysis showed that fragment sizes can be successfully modulated on firefly by changing the bead ratio of the firefly run and no detectable well-to-well contamination was detected by qPCR. Conclusions We have demonstrated that automated bead clean-ups on firefly are uniform show uniformity across both 96- and 384-well plates, with no detectable well-to-well contamination, to deliver the consistent and robust results required for an LDT. Bead-based cleanups on firefly offer a fast, 100% walkaway solution. By running 96-samples at once on firefly, we see approximately a 6-fold increase in throughput than when performed manually by a single user.

B-215 Evaluating RT-qPCR for Dengue Virus Detection: Outcomes of a Brazilian External Quality Assessment Program

Abstract Background Dengue virus (DENV), responsible for a prevalent arthropod-borne viral illness, poses a substantial public health challenge worldwide. Clinically, DENV's manifestation often resembles that of other arboviruses, resulting in frequent healthcare visits and complicating the early diagnosis, which is critical for patient management. While virus isolation is a viable method, RT-qPCR assays yield significantly quicker results. Rapid immunochromatographic tests are available; yet, RT-qPCR not only matches their sensitivity but also provides distinct advantages in clinical laboratory settings. These include higher throughput and integrated, traceable quality control within each routine, offering less subjective and more dependable results. Maintaining high diagnostic standards for DENV is imperative. External quality assessment programs (EQAP) play a key role in enhancing laboratory diagnostic capabilities by monitoring and assessing diagnostic methods. This study evaluates the diagnostic efficacy of RT-qPCR for DENV using data from an EQAP conducted by a Brazilian provider, accredited by ABNT NBR ISO/IEC 17043:2011. Methods The quality control samples comprised lyophilized suspensions containing viable DENV particles, grown on vero cells (BCRJ 0245/ATCC CCL-81) under BSL-3 conditions. From February 2017 to November 2022, twenty-four EQAP survey rounds were carried out, each encompassing three samples, culminating in 1,153 data points. The evaluation metrics included participant numbers, adequacy percentage, sensitivity, specificity, and the incidence of false positives and negatives. The Mann-Kendall test assessed trends in these metrics over the survey rounds. The RT-qPCR kits employed by participants were classified either as in-vitro diagnostic (IVD) or as laboratory-developed tests (LDT). Kits that demonstrated high performance in terms of adequacy, sensitivity, and specificity were identified and listed. Results The number of laboratories participating has increased. Starting with four in 2017, the program expanded to 28 by 2022. Laboratory accuracy was high during the evaluation period, with a median of 99% (97.8% in 2017 and 99.2% in 2022). The Mann-Kendall test indicated a significant trend of increased accuracy (p<0.001). Specificity remained stable at 98.8%, while sensitivity improved, with a median of 99.4%—rising from 93.3% in 2017 to 100% in 2022 (p<0.001). Only three false negatives were reported over three separate rounds, and six false positives were reported across four rounds. Both LDT and IVD RT-qPCR kits demonstrated high accuracy (99.4% and 98.4%, respectively). Biomanguinhos ZDC, Viasure ZDC, and ZDC Multiplex kits all showed 100% adequacy, sensitivity, and specificity. Conclusions This study highlights the increasing adoption of RT-qPCR for DENV detection, as evidenced by the growing number of laboratories in the EQAP program and the consistently high adequacy rates from 2017 to 2022. Nonetheless, the number of participating laboratories remains relatively small for a vast tropical country like Brazil. The high sensitivity and specificity, along with the infrequency of false results, underscore its effectiveness. The superior performance of certain kits suggests possible advancements in DENV diagnosis, contributing to global public health initiatives against this arboviral disease.

B-212 Laboratory Performance in Molecular Detection of Mycobacterium tuberculosis Through Analysis of External Quality Assessment Program Results: WHO-Recommended Tests Outperform Others

Abstract Background In 2022, tuberculosis caused 11,200 deaths in Brazil and 1,300,000 globally. With 87,344 new cases, it remains a leading infectious cause of death, second only to COVID-19. Prompt detection is critical, particularly with the growing resistance to rifampicin and isoniazid, highlighting the need for molecular testing. External Quality Assessment Programs (EQAP) are essential to ensure the accuracy, reliability, and harmonization of laboratory results amid the complexities of M. tuberculosis infection. Here, we evaluate the overall performance of participating laboratories (labs) in three EQAP related to tuberculosis: M. tuberculosis molecular detection (MTB), Rifampicin resistance detection (RIF), and Isoniazid resistance detection (INH). All are organized by a Brazilian provider accredited according to the ABNT NBR ISO/IEC 17043:2011 standard. Methods The EQAP samples were suspensions containing human cells, with or without the addition of cultured, inactivated, and lyophilized M. tuberculosis. Labs received two samples quarterly for testing accuracy, sensitivity, and specificity. We computed these metrics along with participant and round numbers. The Mann-Kendall test assessed accuracy trends over time. Furthermore, kit type accuracies for in-vitro diagnostic (IVD) vs. laboratory-developed tests (LDT), World Health Organization recommended (WHO-recommended) vs. other kits (non-WHO), and methods (qPCR vs. PCR) were compared using chi-square tests and odds-ratio calculations. Results In the MTB EQAP (2018-2022), 63 labs participated over 20 rounds, with 22 (range: 6-48) labs per round on median, achieving 96.6% accuracy (853/883), 97.5% sensitivity (556/570), and 94.9% specificity (297/313). The Mann-Kendall test found no significant accuracy trends over time (τ = -0.91, p = 0.36). IVDs outperformed LDTs with 97.8% (751/768) vs. 87.9% (102/116) accuracy (OR=6.0, 95%CI 3.42-20.9, p<0.0001). WHO-recommended kits were more accurate than non-WHO kits, 98.2% (674/686) vs. 90.4% (179/198) (OR=5.96, 95%CI 1.05-12.7, p<0.0001). qPCR exceeded the accuracy of PCR, 97.2% (833/857) versus 74% (20/27) (OR=12.1, 95%CI 4.8-32.9, p<0.0001). For RIF EQAP (2020-2022), 49 laboratories in 9 rounds, with 25 (range: 18-42) laboratories per round on median, showed 97.4% accuracy (297/305), 97.2% sensitivity (176/181), and 97.6% specificity (121/124). In the INH EQAP (2021-2022), 7 laboratories over 7 rounds, with 4 (range: 2-5) laboratories per round on median, reached 95.2% accuracy (20/21), 100% sensitivity (4/4), and 94.1% specificity (16/17). Differences between types of kits and methods for RIF and INH, as well as trends over time, were not significant or could not be calculated due to the limited number of samples. Conclusions The MTB EQAP underscored the high participation and suitability of labs, along with excellent diagnostic performance; IVD kits were 6 times more likely to yield adequate results than LDTs. WHO-recommended kits were 5.9 times more likely to produce adequate results than non-WHO kits. qPCR was 12.6 times more likely to achieve adequate results than PCR. Despite fewer participants in RIF and INH, their performance rates were high; more data are needed for conclusive results on kit and method types. This study affirms the quality of molecular diagnostics for tuberculosis in Brazil, informs on kit and method selection, and underscores the critical role of EQAPs in combating a significant global health issue.

A-247 Performance and Compatibility Assessment of Serially Diluted Microbix QAPTM Controls with Seegene NovaplexTM Assays

Abstract Background Choosing high-quality external (third-party) controls like Microbix QAPTM PROCEEDxTM Controls and REDxTM Controls is crucial during test development, validation of Laboratory Developed Tests (LDTs), performance verification, proficiency testing, instrument qualifications, operator training and R&D testing. Laboratories may prefer to include multiple positive controls at varying concentrations to achieve reliable performance across a broad range of detection concentrations. In this study, we assess the performance and compatibility of Microbix QAPTM PROCEEDxTM liquid Controls and REDxTM swabs with Seegene’s real time qPCR assays that utilize TOCE and MuDTTM technologies. TOCE and MuDTTM technologies achieve multiple reads in a single channel and are the backbone of Seegene’s wide coverage panel assays. Methods The controls below were reconstituted and/or diluted in Copan UTM and extracted on the Seegene STARlet liquid handler using Seegene’s STARmag Universal extraction kit. After extraction, PCR was run on the Bio-Rad CFX 96 Opus Dx and analyzed using the Seegene Viewer software. Results Microbix QAPsTM liquid formulations were tested at various dilutions from stock up to 1/2048 with N=5per dilution. The VP-13-01 Flu A control was detected up to a concentration of1/2048 and at this dilution had Ct values of 30 + 0.3 for the Novaplex Respiratory Panel 1A (RP1A) Assay, 28.6 + 0.3 cycles for the Seegene SARS-CoV-2/FluA/FluB/RSV (Quad) Assay, and 27.9 + 0.3 for the Respiratory Virus Master (RVM) Assay. The Microbix VP-14-01 Flu B control was detected up to 1/2048 with Ct values of 34.3 + 0.3 for the RP1A Assay, 30 + 0.5 for the Quad Assay, and 30.3 + 0.3 for the RVM Assay. The VP-07-01 RSV A control was also detected at a 1/2048 dilution with Ct values of 32.7 + 0.3 for RP1A, 30.6 + 0.6 for Quad Assay, and 29.2 + 0.4 for RVM. The VP-61-01 Trichomonas vaginalis (TV) control was detected up to a dilution of 1/128 with a Ct value of 37.3 + 0.4 when tested with the Seegene Sexually Transmitted Infection Essential (STI) Assay. The VP-02-M1 HSV1 control was detected up to a dilution of 1/128 with a Ct value of 31.3 + 0.7 for the Seegene Genital Ulcer (GU) Assay. The VP-23-M1 HSV2 control was detected p to a dilution of 1/128 with a Ct value of 31.9 + 0.5 for the GU Assay. Microbix also makes a dry swab formulation product line that we tested in 3mL of UTM. Of these we tested the RED-S-10-M1 swab covering the SARS-CoV-2, Flu A, Flu B, and RSV controls. The VP-S-12-M5 swab covering the Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG), and TV controls. We also tested the VP-S-03-M1 swab covering HSV1, HSV2, VZV and syphilis controls. When tested with various assays all swabs were detected at the stock formulation. Conclusions Microbix QAPsTM are inactivated whole-organism controls, mimicking patient samples, and meet the stringent demand for quality or performance assessments. Their compatibility with Seegene’s Assays over a range of dilutions and formulations makes them ideal for ensuring reliable detection across multiple diagnostic needs.

B-103 Correlation of Novel Markers, Anti-Sa (Citrullinated Vimentin) and Anti-CarP (Carbamylated Protein), with High Vectra® DA Disease Activity Scores in Rheumatoid Arthritis (RA)

Abstract Background Citrullination and carbamylation are post-translational modifications where autoantibodies against citrullinated and carbamylated proteins in RA have been associated with more aggressive disease. At the same time, Vectra® DA is blood test that yields a RA disease activity score from 1 to 100. Here, 120 patient samples were analyzed to assess the relationship of disease activity as measured by Vectra® score and the presence of Anti-Sa (Citrullinated Vimentin) and Anti-CarP (Carbamylated Protein). Methods Serum samples were analyzed for Vectra® DA (which is validated for adults with RA) where 12 biomarker measurements (VCAM-1, EGF, VEGF-A, IL-6, TNF-RI, MMP-1, YKL-40, Leptin, Resistin, SAA, CRP) together with patient age, gender and adiposity are used to generate a score where established categories are low for 1 to 29, moderate for 30 to 44, and high for 45 to 100. Anti-Sa and Anti-CarP were by ELISA lab-developed tests (LDT) with a cut-off for positive of >20 Units (U). Results Anti-Sa and Anti-CarP for 40 samples from each Vectra® DA category are reported. Patients with the highest Vectra® scores (mean 58.2, range 46.8 - 87.5) had highest rates of positivity for Anti-Sa (52.5%) and Anti-CarP (35%). In contrast, the group with the lowest Vectra® scores (mean 11.8, range 3.8 - 18.1) were infrequently positive for Anti-Sa (17.5%) and Anti-CarP (0%). Frequencies with moderate Vectra® scores (mean 37.1, range 32.4 - 41.8) were mid-range: Anti-Sa (25%) and 7.5% for Anti-CarP (7.5%). Of note, positivity of Anti-Sa was analytically stronger, i.e. higher titer, in patients with moderate or high Vectra® (mean 90.0U or 81.7U), compared to values seen in those with low Vectra® (mean 60.4 U). Conclusions Our analysis of 120 patient samples demonstrates that high disease activity as determined by Vectra® DA corresponds to higher positivity of novel autoantibody markers, Anti-Sa and Anti-CarP. High Vectra® DA scores (>44) are associated with a 20% 1-year risk of radiographic progression while low scores (<30) carry only a 1% risk. Here, we found that patients with high Vectra® scores were 3 times more likely to test positive for Anti-Sa than those with low disease activity scores. At the same time, Anti-CarP occurred in high frequency (35%) with high Vectra® in contrast to zero positivity observed with low Vectra® scores. Thus, our findings support Anti-CarP’s reported association with more active disease with higher risk of developing joint erosions. Also of note, patients with high Vectra® scores had particularly high Anti-Sa positivity - both a 52.5% positivity rate as well as higher Anti-Sa levels (titers) than the group with lower Vectra® scores. These data corroborate other studies where Anti-Sa not only predicted more aggressive, rapid disease progression, but its titers correlated with clinical measures of disease activity. Taken together, our data support the consistency and clinical usefulness of the Vectra® score along with novel autoantibody markers, Anti- Anti-Sa and Anti-CarP, in order to assess RA severity, prognosis, and patient-specific treatment strategies.

So I Guess I Am a Device? Making the Case for Professional Practice in the Context of the FDA’s Final Rule on Laboratory-Developed Tests

So I Guess I Am a Device? Making the Case for Professional Practice in the Context of the FDA’s Final Rule on Laboratory-Developed Tests

IVDCheckR- simplifying documentation for laboratory developed tests according to IVDR requirements by introducing a new digital tool.

A recent challenge for clinical laboratories is the lack of clear guidelines for handling significant modifications of CE-marked assays. The modifications may involve, for example, extending measurement intervals, changing dilution procedures or using non-validated sample materials. The challenge arises due to the amended Regulation (EU) 2017/746 on invitro diagnostic medical devices (IVDR), which is now poised for implementation, despite the extended transition periods. The IVDR application imposes challenges not only for diagnostic companies but also for clinical laboratories when using laboratory developed tests (LDTs), often referred to as in-house assays. In this context, a coherent and meticulously structured LDT documentation is highly beneficial. While laboratories are obliged to meet the IVDR requirements, the absence of a streamlined framework or guideline hampers the ability to gain a comprehensive overview on the requirements and possible options for their fulfilment. To address this issue, we introduce a web based digital tool powered by an R Shiny web application. This tool facilitates a seamless implementation of IVDR requirements for LDTs across diverse laboratory environments in terms of their transparency and validity. Our approach focuses on adequate handling of significant modifications of CE-marked invitro diagnostic medical devices (IVD). IVDRCheckR is an open-source tool that is easily accessible and free from system dependencies. The tool promotes a seamless process and a guide to enhance transparency, reliability, and validity of laboratory examination results based on LDTs. Additionally, the tool further provides modules for evaluating quality control data and quantitative method comparison data. Our Shiny web application-based platform is adigitised, user-friendly tool that simplifies the documentation for LDTs according to IVDR requirements with special emphasis on solutions for handling modifications to CE-marked assays.

The Supreme Court’s Loper Bright Ruling

This Viewpoint explores the US Supreme Court’s ruling in Loper Bright Enterprises v Raimondo and the implications the ruling could have on the regulation of laboratory-developed tests and public health.

Evaluation of a Benzodiazepine Immunoassay for Urine Drug Testing in Clinical Specimens.

Benzodiazepines are commonly prescribed medications frequently linked to instances of abuse and overdose. Historically, FDA-cleared benzodiazepine urine immunoassays cross-react poorly with glucuronidated benzodiazepine metabolites, leading to false negatives. Clinical laboratories have addressed this deficiency by creating laboratory-developed tests (LDTs) that incorporate a beta-glucuronidase hydrolysis step to increase the clinical sensitivity of these assays. Performance characteristics of 2 FDA-cleared benzodiazepine urine immunoassays (Benzodiazepines Plus, no glucuronidase and Benzodiazepines II, with glucuronidase; Roche Diagnostics) and a previously described benzodiazepine immunoassay LDT (with glucuronidase) were evaluated using 258 clinical urine specimens. The positive immunoassay cutoff was set at 200 ng/mL of nordiazepam and results were compared to an LC-MS/MS benzodiazepine LDT. Clinical sensitivity, specificity, precision, and immunoassay cross-reactivity were determined for all 3 immunoassays. The Benzodiazepines II and LDT immunoassays exhibited greater clinical sensitivity (100% and 95.2%) compared to the Benzodiazepines Plus assay (66.7%). Clinical specificity of 100% was observed for all 3 assays. Immunoassay response of the Benzodiazepines II assay was greater across the range of concentrations tested (100-1000 ng/mL) relative to the other immunoassays and was the most sensitive immunoassay for the detection of lorazepam glucuronide. The Benzodiazepines II immunoassay demonstrated the greatest clinical and analytical sensitivity compared to the Benzodiazepines Plus and LDT immunoassays. The incorporation of beta-glucuronidase was crucial, as the Benzodiazepines II and LDT immunoassays demonstrated superior clinical sensitivity when compared to the Benzodiazepines Plus immunoassay that does not incorporate a beta-glucuronidase hydrolysis step.

Patient safety and healthcare quality of U.S. laboratory developed tests (LDTs) in the AI/ML era of precision medicine.

This policy brief summarizes current U.S. regulatory considerations for ensuring patient safety and health care quality of genetic/genomic test information for precision medicine in the era of artificial intelligence/machine learning (AI/ML). The critical role of innovative and efficient laboratory developed tests (LDTs) in providing accurate diagnostic genetic/genomic information for U.S. patient- and family-centered healthcare decision-making is significant. However, many LDTs are not fully vetted for sufficient analytic and clinical validity via current FDA and CMS regulatory oversight pathways. The U.S. Centers for Disease Control and Prevention's Policy Analytical Framework Tool was used to identify the issue, perform a high-level policy analysis, and develop overview recommendations for a bipartisan healthcare policy reform strategy acceptable to diverse precision and systems medicine stakeholders.

Rule Expansion Further Regulates Laboratory Developed Tests.

Rule Expansion Further Regulates Laboratory Developed Tests.

Infrastructure Limitations of U.S. Food and Drug Administration Proposed Review of Laboratory-Developed Tests.

Infrastructure Limitations of U.S. Food and Drug Administration Proposed Review of Laboratory-Developed Tests.

An update on the status of direct testing for Treponema pallidum subspecies pallidum for the laboratory diagnosis of syphilis in Canada

Background: In Canada, the recent resurgence of infectious syphilis and rising rates of congenital syphilis have renewed interest in direct detection methods for the laboratory diagnosis of syphilis. The Canadian Public Health Laboratory Network (CPHLN) has previously published a series of guidelines for the diagnosis of syphilis in Canada, including the use of direct tests. In the decade since those guidelines were published, laboratory practice has changed. Methods: This systematized review combined a literature search (MEDLINE) of methods for direct detection of Treponema pallidum with an informal survey of current testing practices in Canadian public health laboratories. Results: Direct testing methods have favourable performance characteristics for detection of early syphilis and congenital cases. Although no government licensed commercial nucleic acid amplification test (NAAT) for syphilis is available in Canada, laboratory-developed tests have been implemented in multiple Canadian jurisdictions. Clinical specimens with the highest yield of positive NAAT results for syphilis include genital ulcers, skin lesions, and oral swabs from primary and secondary syphilis patients. For investigation of congenital syphilis, nasopharyngeal, placenta, umbilical cord, blood, and skin lesions are specimens of choice for direct detection of T. pallidum by NAAT. Conclusions: This update on the status of direct testing highlights the importance of NAAT for the detection of T. pallidum, the reduced role of microscopy-based methods, and the emergence of DNA and genome sequencing as tools for phylogenetic analysis and molecular epidemiology.

FDA's Final Rule on Laboratory Developed Tests.

FDA's Final Rule on Laboratory Developed Tests.

Utilization of multiplex molecular panels for urinary tract infections, Medicare claims, 2016 – 2022

Background: Multiplex molecular tests for infectious diseases can provide highly sensitive results rapidly; however, these tests may more readily detect asymptomatic colonization. There are reports of non-FDA approved laboratory-developed multiplex tests for the diagnosis of urinary tract infections (UTI). Differentiating UTI from asymptomatic bacteriuria is challenging, especially in older adults. The increased sensitivity of multiplex tests may exacerbate this challenge. We sought to describe the use of multiplex testing for UTIs in Medicare claims. Methods: Multiplex testing was identified using carrier claims submitted by non-institutional providers using the Chronic Conditions Warehouse for 2016 – 2022. Because there are no CPT-4 codes specifying UTI multiplex testing, we included claims as described in Figure 1 and categorized claims based on the primary ICD-10-CM diagnosis. The payment amounts for line items related to testing for infectious agents were summed. Laboratories were counted using CLIA numbers listed on corresponding claims. Beneficiaries residing in a nursing home at the time of their claim were identified using stay information derived from the Minimum Dataset 3.0. For comparison, similar characteristics among carrier claims with a CPT-4 code indicating urine culture were also described. Results: Claims for unspecified multiplex molecular tests overall have increased, driven by increases in claims with a primary UTI diagnosis (from 8,521 in 2016 to 386,943 in 2022), while urine cultures have not (Figure 1). In 2022, 65% of all unspecified multiplex tests were linked to a diagnosis of UTI; UTI multiplex claims were associated with 647 laboratories. For UTI claims, the median cost per claim for line items related to multiplex testing was $589 compared to $13 for urine culture-related line items. Overall, 8% of UTI multiplex claims were for beneficiaries residing in a nursing home. Conclusions: Claims for non-FDA approved unspecified multiplex tests associated with a primary diagnosis of UTI have increased >45-times between 2016-2021 and have >45-times higher median costs than urine cultures. The use of this testing in the Medicare population, including nursing home residents, is of potential concern given that inappropriate treatment of asymptomatic bacteriuria has been described to be common in older adults. Research is needed to outline use cases where UTI multiplex testing may be beneficial. Appropriate use of diagnostic testing is important to minimize diagnostic errors and avoid unnecessary antibiotic use.