First-line Test Research Articles

ESR Essentials: imaging in stable chest pain - practice recommendations by ESCR.

Stable chest pain is a common symptom with multiple potential causes. Non-invasive imaging has an important role in diagnosis and guiding management through the assessment of coronary stenoses, atherosclerotic plaque, myocardial ischaemia or infarction, and cardiac function. Computed tomography (CT) provides the anatomical evaluation of coronary artery disease (CAD) with the assessment of stenosis, plaque type and plaque burden, with additional functional information available from CT fractional flow reserve (FFR) or CT myocardial perfusion imaging. Stress magnetic resonance imaging, nuclear stress myocardial perfusion imaging, and stress echocardiography can assess myocardial ischaemia and other cardiac functional parameters. Coronary CT angiography can be used as a first-line test for many patients with stable chest pain, particularly those with low to intermediate pre-test probability. Functional testing may be considered for patients with known CAD, where the clinical significance is uncertain based on anatomical testing, or in patients with high pre-test probability. This practice recommendations document can be used to guide the selection of non-invasive imaging for patients with stable chest pain and provides brief recommendations on how to perform and report these diagnostic tests.

Abstract LB095: ASPYRE-Lung addresses critical gaps in NGS-based biomarker testing: Robust variant calling from NGS QC fails

Abstract Advanced NSCLC treatment guidelines recommend testing patients for genomic biomarkers to utilize over 30 FDA-approved targeted therapies. A major challenge with current NGS-based testing methods is that ~45% of patient samples fail NGS quality control (QC), leading to inadequate patient care. The ASPYRE® technology addresses the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants. The ASPYRE-Lung® panel covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management, based on clinical practice guidelines. The assay concurrently detects single nucleotide variants, insertions, deletions, and gene fusions from NSCLC tissue-derived DNA and RNA. In this study, we investigated an NSCLC patient sample set from Precision for Medicine, including biobanked clinical specimens that had previously failed NGS QC, and for which genomic biomarker data were unavailable. 120 matched DNA and RNA biobanked NSCLC FFPE patient samples previously analyzed by Illumina TSO 500 were tested using ASPYRE-Lung in Biofidelity’s CLIA laboratory, including 94 samples which failed NGS QC parameters for DNA, RNA, or both, and 26 control samples which passed NGS QC. Samples were tested by ASPYRE-Lung using an input of 20 ng DNA and 6 ng RNA, except for 5 patient samples that were run at lower inputs, ranging from 4.25 to 14 ng DNA. Key findings:•Of the 94 patient samples that failed NGS QC, 98% of samples (92/94) passed ASPYRE-Lung QC and could inform patient care.•In the 92 patient samples that passed ASPYRE-Lung QC, 47% (43/92) had a detectable variant identified by ASPYRE-Lung.•All 26 control samples that passed NGS QC also passed ASPYRE-Lung QC.•Of the 5 samples with 4.25 to 14 ng DNA inputs to ASPYRE-Lung, data were generated on 4/5 samples and 2/4 had a detectable variant.•ASPYRE-Lung has the potential to provide actionable clinical information on patient samples deemed to be of insufficient quantity for NGS testing.ASPYRE-Lung has a high success rate, is easily adoptable and cost effective making it suitable as a first-line testing option, or for samples that are either QNS or fail NGS QC, and can provide a large fraction of NSCLC patients with actionable biomarker information, with potentially smaller tissue requirements. ASPYRE-Lung genomic testing is transformative for cancer care management, and allows more patients with NSCLC to benefit from effective and better tolerated therapies. Citation Format: Ryan T. Evans, Elyse Shapiro, Elizabeth Gillon-Zhang, Julia N. Brown, Candace King, Cory Kiser, Mary Beth Rossi, Darren Davis, Michelle L. Taylor, Jennifer Clower, Jian Yang, Cullen A. Taylor, Robert W. Snyder, Wendy J. Levin. ASPYRE-Lung addresses critical gaps in NGS-based biomarker testing: Robust variant calling from NGS QC fails [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB095.

Prospective Evaluation of a New Liquid-Type Rapid Urease Test Kit for Diagnosis of Helicobacter pylori.

Rapid and accurate diagnostic tools are essential for the timely recognition of Helicobacter pylori (H. pylori) in clinical practice. The rapid urease test (RUT) is a comparatively accurate and time-saving method recommended as a first-line diagnostic test. The primary objective of conducting the RUT is to obtain rapid results, thus enabling the initiation of an eradication therapy based on clarithromycin resistance testing. This study aimed to assess the reaction time and accuracy of a new liquid-type RUT. In this prospective study, consecutive dyspeptic or check-up patients referred to our clinic for endoscopy were assessed to evaluate the rapidity and accuracy of a novel liquid-type RUT (Helicotest®, WON Medical, Bucheon, Republic of Korea) compared with another commercial RUT kit (HP kit, Chong Kun Dang, Seoul, Republic of Korea) and a real-time quantitative PCR-based assay (Seeplex® H.pylori-ClaR Detection, Seegene, Republic of Korea). RUTs were analyzed at 10 min, 30 min, 60 min, and 120 min. Among the 177 enrolled patients, 38.6% were infected with H. pylori. The positivity rates of the liquid-type RUT were 26.1, 35.8, 39.2%, and 41.5% at 10, 30, 60, and 120 min, respectively. When compared with the HP kit test, the time needed to confirm positivity was significantly reduced by 28.6 min (95% CI, 16.60-39.73, p < 0.0001). Helicotest® had a greater accuracy (96.02 ± 1.47), sensitivity (98.53 ± 1.46) and NPV (99.03 ± 0.97) compared to the HP kit. Compared to the commonly used RUT, the new liquid-type RUT presented faster and reliable results. Such findings could improve H. pylori treatment outcomes, particularly in outpatient clinical settings.

Accessibility, availability and common practices regarding genetic testing for epilepsy across Europe: A survey of the European Reference Network EpiCARE.

The increasingly rapid pace of advancement in genetic testing may lead to inequalities in technical and human resources with a negative impact on optimal epilepsy clinical practice. In this view, the European Reference Network (ERN) for Rare and Complex Epilepsies EpiCARE conducted a survey addressing several aspects of accessibility, availability, costs, and standard practices on genetic testing across ERN EpiCARE centers. An online Google form was sent to 70 representatives of ERN EpiCARE centers. Descriptive statistics and qualitative analysis were used for data presentation. We received 45 responses (1/center) representing 23 European countries with a better representation of Western Europe. Forty-five percent of the centers did not have access to all available types of genetic testing, mainly reflecting the limited availability of whole-genome sequencing (WGS). Thirty-five percent of centers report cost coverage only for some of the available tests, while costs per test varied significantly (interquartile range IQR ranging from 150 to 1173 euros per test across centers). Urgent genetic testing is available in 71.7% of countries (time-to-urgent result: 2 day to 2 months). The average time-to-result of specific tests in case of non-urgent prescription has a significant variance across centers, with the biggest range observed for whole-exome sequencing (6-128 weeks, IQR: 27 weeks). The percentage of agreement among the experts regarding the choice of genetic test at first intention in specific clinical situations was in all cases less than 50 percent (34.9% to 47% according to the proposed scenarios). Costs, time to deliver the results to the clinician, and type of first-line genetic testing vary widely across Europe, even in countries where ERN EpiCARE centers are present. Increased availability of genetic tests and guidance for optimal test choices in epilepsy remains essential to avoid diagnostic delays and excess health costs. The survey of the ERN EpiCARE highlights disparities in genetic testing for epilepsy across 45 ERN EpiCARE centers in 23 European countries. The findings reveal variable access to certain genetic tests, with lowest access to WGS. Costs and time-to-results vary widely. Urgent genetic testing is available in 71.7% of countries. Agreement among experts on first-line genetic tests for specific patient scenarios is below 50%. The study emphasizes the need for improved test availability and guidance to avoid diagnostic delays and unnecessary costs. EpiCARE has the mission to contribute in homogenizing best practices across Europe.

Quantification of Hepatitis E Virus ORF2 Protein by a Novel Sandwich ELISA.

Hepatitis E virus (HEV) causes acute hepatitis in humans, which can progress to chronicity in immunosuppressed individuals. Almost all reported HEV infections are caused by Paslahepevirus balayani genotypes 1-4. The structural ORF2 protein is the major antigen detected in the blood of HEV-infected individuals. ELISA assays to detect IgM antibodies to HEV are the first-line diagnostic tests; however, they showed variable performance with frequently discordant results. A qualitative HEV antigen (ORF2) ELISA is currently available for research use. Here, we report a novel quantitative sandwich ELISA to measure HEV ORF2 protein in 3 matrix types. An optimal pair of capture and detection antibodies was selected among 12 unique combinations tested. A sandwich ELISA protocol was developed using these mAbs and biotin-streptavidin technology. The protocol was further optimized to quantify ORF2 antigen in different matrices by interpolating from a standard curve with a linear range of 3.17 to 50.8 femtomoles/mL. Using this method, ORF2 protein was detected in the cell culture medium of Huh7 cells as early as 2-3 days after transfection with HEV genome RNA and in a medium of human hepatocytes infected with HEV. ORF2 antigen was readily detected in the first 2 weeks post-HEV infection in gerbil sera. In immunosuppressed gerbils, ORF2 was detected up to 6 weeks, and the levels were significantly higher between 3 and 6 weeks post-infection. HEV ORF2 antigen levels showed a strong positive correlation with HEV RNA levels in both cell culture medium and gerbil sera. Our novel sandwich ELISA detected at least 7.3 femtomoles/mL ORF2 protein in human plasma spiked with cell culture propagated HEV and detected ORF2 protein in human plasma samples that tested positive for HEV RNA but negative for anti-HEV antibodies. Further, the assay was nonreactive, with negative human plasma, and HBV or HCV-positive human plasma demonstrating specificity. Overall, our ORF2 antigen ELISA will be useful for quantifying ORF2 antigen in cell culture medium, gerbil serum, and human plasma. Further studies are warranted to evaluate its utility in HEV clinical diagnosis.

Sensitivity and Specificity Analysis of Fine Needle Aspiration Cytology (FNAC) for Thyroid Swelling.

Introduction Thyroid gland disorders, such as goiters or tumor masses, are the result of localized or systemic aberrations of the thyroid gland. The purpose of this research was to see how effective fine needle aspiration cytology (FNAC) was in detecting thyroid swelling in patients with thyroid swelling. It is critical to be able to differentiate between benign and malignant thyroid nodules to reduce unnecessary thyroid surgeries. It is hypothesized that FNAC is not a reliable diagnostic tool to detect thyroid nodules in patients with thyroid swelling and that there is a significant variation between the number of thyroid nodules detected by FNAC and the number of thyroid nodules that were eventually diagnosed as malignant by surgery. The significance of this research shows the effectiveness of diagnostic tests for thyroid nodules in patients with thyroid swelling can help reduce unnecessary surgeries and improve patient care. Methods This cross-sectional study was carried out at the Department of Pathology at a tertiary care hospital in central India, from March 1, 2022 to June 31, 2022. Fifty patients with thyroid swelling were covered in the study. All patients were chosen after an ultrasound revealed goiter on clinical grounds. Results The majority of patients in the age group 31-40 years (33.33%) were female (74.67%). About 54.67% of the patients had a single thyroid nodule. FNAC had a sensitivity of 95.38%, a specificity of 53.33%, a positive predictive value of 86.67% and a negative predictive value of 88.57%. Conclusion FNAC is a simple, economical, and commonly used first-line diagnostic method for thyroid cancer. A false negative or false positive cytological diagnosis may be produced as a result of thyroid cytology. The study emphasizes the need to improve basic healthcare in rural India by treating FNAC as a first-line diagnostic test for thyroid swellings to guide management, although it does not replace histopathology.

A comparison of the performance of 68Ga-Pentixafor PET/CT versus adrenal vein sampling for subtype diagnosis in primary aldosteronism.

To investigate the diagnostic efficiency and prognostic value of 68Ga-Pentixafor PET/CT in comparison with adrenal vein sampling (AVS) for functional lateralization in primary aldosteronism (PA). Histology and long-term clinical follow-up normally serve as the gold standard for such diagnosis. We prospectively recruited 26 patients diagnosed with PA. All patients underwent 68Ga-Pentixafor PET/CT and AVS. Postsurgical biochemical and clinical outcomes of patients with unilateral primary aldosteronism (UPA), as diagnosed by PET/CT or AVS, were assessed by applying standardized Primary Aldosteronism Surgical Outcome (PASO) criteria. Immunohistochemistry (IHC) was performed to detect the expression of aldosterone synthase (CYP11B2) and CXCR4. On total, 19 patients were diagnosed with UPA; of these, 13 patients were lateralized by both PET/CT and AVS, four patients were lateralized by PET-only, and two by AVS-only. Seven subjects with no lateralization on AVS and PET received medical therapy. All patients achieved complete biochemical success except one with nodular hyperplasia lateralized by AVS alone. The consistency between PET/CT and AVS outcomes was 77% (20/26). Moreover, CYP11B2-positive nodules were all CXCR4-positive and showed positive findings on PET. Patients who achieved complete biochemical and clinical success had a higher uptake on PET as well as stronger expression levels of CXCR4 and CYP11B2. Our analysis showed that 68Ga-Pentixafor PET/CT could enable non-invasive diagnosis in most patients with PA and identify additional cases of unilateral and surgically curable PA which could not be classified by AVS. 68Ga-Pentixafor PET/CT should be considered as a first-line test for the future classification of PA.

Ultrahigh-Spatial-Resolution Photon-counting Detector CT Angiography of Coronary Artery Disease for Stenosis Assessment.

Background Coronary CT angiography is a first-line test in coronary artery disease but is limited by severe calcifications. Photon-counting-detector (PCD) CT improves spatial resolution. Purpose To investigate the effect of improved spatial resolution on coronary stenosis assessment and reclassification. Materials and Methods Coronary stenoses were evaluated prospectively in a vessel phantom (in vitro) containing two stenoses (25%, 50%), and retrospectively in patients (in vivo) who underwent ultrahigh-spatial-resolution cardiac PCD CT (from July 2022 to April 2023). Images were reconstructed at standard resolution (section thickness, 0.6 mm; increment, 0.4 mm; Bv44 kernel), high spatial resolution (section thickness, 0.4 mm; increment, 0.2 mm; Bv44 kernel), and ultrahigh spatial resolution (section thickness, 0.2; increment, 0.1 mm; Bv64 kernel). Percentages of diameter stenosis (DS) were compared between reconstructions. In vitro values were compared with the manufacturer specifications of the phantom and patient results were assessed regarding effects on Coronary Artery Disease Reporting and Data System (CAD-RADS) reclassification. Results The in vivo sample included 114 patients (mean age, 68 years ± 9 [SD]; 71 male patients). In vitro percentage DS measurements were more accurate with increasing spatial resolution for both 25% and 50% stenoses (mean bias for standard resolution, high spatial resolution, and ultrahigh spatial resolution, respectively: 10.1%, 8.0%, and 2.3%; P < .001). In vivo results confirmed decreasing median percentage DS with increasing spatial resolution for calcified stenoses (n = 161) (standard resolution, high spatial resolution, and ultrahigh spatial resolution, respectively: 41.5% [IQR, 27.3%-58.2%], 34.8% [IQR, 23.7%-55.1%], and 26.7% [IQR, 18.6%-44.3%]; P < .001), whereas noncalcified (n = 13) and mixed plaques (n = 19) did not show evidence of a difference (P ≥ .88). Ultrahigh-spatial-resolution reconstructions led to reclassification of 62 of 114 (54.4%) patients to lower CAD-RADS category than that assigned using standard resolution. Conclusion In vivo and in vitro coronary stenosis assessment improved for calcified stenoses by using ultrahigh-spatial-resolution PCD CT reconstructions, leading to lower percentage DS compared with standard resolution and clinically relevant rates of reclassification. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by McCollough in this issue.

Use of video-electroencephalography as a first-line examination in veterinary neurology: development and standardization of electroencephalography in unsedated dogs and cats.

To assess the feasibility and validate the use of video-electroencephalography (EEG) in conscious dogs and cats and to propose guidelines of routine EEG in veterinary clinical practice. Prospective clinical study. One hundred and fifty EEG recordings were carried out to validate the clinical adding-value, reproducibility, and guidelines on 140 owned animals. One hundred and one EEGs were performed on dogs and 49 on cats. We compared recordings performed with 8 EEG unwired stud Ag/AgCl electrodes held by elastic straps and 8 EEG wired cup Ag electrodes held by a tailor-made manufactured headset combined with a wired video-EEG device. Electrodes placement was determined according to previously published animal EEG protocols. Physiological sensors, such as electrocardiography, electromyography, and respiratory sensors were added. Stimulation protocols were tested. Quality and interpretability were evaluated. Headsets and recording procedures appeared suitable for all skull shapes and sizes. Video-EEG recordings were successfully performed without tranquilization or anesthesia except for 9 animals. Median EEG recordings time was 40 min. Impedance remained below 20 kΩ in 99% of dog EEGs and 98% of cat EEGs. Isosynchrony was reported in 6% of the channels. Seventy-five percent of dog EEGs and 83% of cat EEGs were readable for more than 50% (to 100%) of their duration. Successful discrimination of vigilance states from rhythm analysis (wakefulness, drowsiness, and sleepiness) was possible in 99% of dog EEGs and 91% of cat EEGs. Photic driving responses during photic stimulations were observed in 11% of dog EEGs and 85% of cat EEGs. Electroencephalography recordings were directly informative in 32% of the examinations: in 25% EEG abnormalities were associated with clinical signs and 7% concerned EEG abnormalities without clinical symptoms during recording. Thirteen percent of dogs subjected to photic stimulation exhibited epileptic anomalies. Among 9 EEGs with other history-based stimulations, three displayed epileptic graphoelements. We have developed a standardized unanesthetized video-EEG procedure easily performed and reproducible in dogs and cats. Qualitative and quantitative technical and medical criteria were evaluated and were in accordance with human EEG recommendations. Moreover, we have demonstrated its relevance and accuracy for diagnostic purposes, providing further arguments for the use of EEG as a first-line neurological functional exploration test.

Lessons Learned from Translating Genome Sequencing to Clinical Routine: Understanding the Accuracy of a Diagnostic Pipeline.

The potential of genome sequencing (GS), which allows detection of almost all types of genetic variation across nearly the entire genome of an individual, greatly expands the possibility for diagnosing genetic disorders. The opportunities provided with this single test are enticing to researchers and clinicians worldwide for human genetic research as well as clinical application. Multiple studies have highlighted the advantages of GS for genetic variant discovery, emphasizing its added value for routine clinical use. We have implemented GS as first-line genetic testing for patients with rare diseases. Here, we report on our experiences in establishing GS as a reliable diagnostic method for almost all types of genetic disorders, from validating diagnostic accuracy of sequencing pipelines to clinical implementation in routine practice.

Whole Exome Sequencing as a First-Line Molecular Genetic Test in Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathies (DEE) are severe neurodevelopmental disorders characterized by recurrent, usually early-onset, epileptic seizures accompanied by developmental impairment often related to both underlying genetic etiology and abnormal epileptiform activity. Today, next-generation sequencing technologies (NGS) allow us to sequence large portions of DNA quickly and with low costs. The aim of this study is to evaluate the use of whole-exome sequencing (WES) as a first-line molecular genetic test in a sample of subjects with DEEs characterized by early-onset drug-resistant epilepsies, associated with global developmental delay and/or intellectual disability (ID). We performed 82 WESs, identifying 35 pathogenic variants with a detection rate of 43%. The identified variants were highlighted on 29 different genes including, 3 new candidate genes (KCNC2, STXBP6, DHRS9) for DEEs never identified before. In total, 23 out of 35 (66%) de novo variants were identified. The most frequently identified type of inheritance was autosomal dominant de novo (60%) followed by autosomal recessive in homozygosity (17%) and heterozygosity (11%), autosomal dominant inherited from parental mosaicism (6%) and X-linked dominant de novo (6%). The most frequent mutations identified were missense (75%) followed by frameshift deletions (16%), frameshift duplications (5%), and splicing mutations (3%). Considering the results obtained in the present study we support the use of WES as a form of first-line molecular genetic testing in DEEs.

ВЕРИФІКАЦІЯ АЛГОРИТМІВ ТЕСТУВАННЯ НА ВІЛ З ВИКОРИСТАННЯМ ШВИДКИХ ТЕСТІВ

Ensuring accurate, timely and accessible diagnosis of HIV infection is a priority measure aimed at achieving global goals set for Ukraine to overcome the HIV epidemic, namely obtaining quality so that 95% of people living with HIV know their status. Timely diagnosis of HIV infection with the help research results of guaranteed of rapid tests (RT) becomes especially important in the conditions of martial law, when access to ELISA testing is limited or absent at all. A standardized testing strategy and the use of quality-assured products are extremely important for accurate diagnosis, and the wrong choice of testing algorithm can lead to diagnostic errors. WHO Consolidated guidelines on HIV testing services (2019 edition) recommends a single «standard» HIV testing strategy for all HIV testing facilities — three consecutive positive HIV serological test results and a simplified case analysis scheme for testing when discrepant test results are obtained. The causes of misdiagnosis are multifactorial and include errors at various stages of the testing process, but the main factor in the occurrence of a significant number of misdiagnoses is the incorrect selection of tests and/or the sequence of their use. Verification of testing algorithms makes it possible to obtain objective factual data on the effectiveness of a certain combination of tests for accurate diagnosis of HIV infection before their mass introduction. For this, during verification, the selected products are checked for the absence of the same false results, which could lead to incorrect diagnosis. Therefore, an urgent issue for the country is the need to define a reliable national HIV testing algorithm with a backup algorithm, which would be used as an alternative in cases when, for any reason, it is temporarily impossible to conduct research according to the main algorithm. The purpose of the work. Based on the assessment of specificity, to determine the best combinations of RTs, the joint use of which will ensure obtaining guaranteed test results (with the smallest or zero false cross-reactivity) within the framework of the selected testing algorithms. Materials and methods. This verification study was based on an HIV testing strategy using three RTs sequentially, the first of which should be the most sensitive (≥99% for both true and false-reacitive results); the second and third tests should be the most specific (≥98% to detect all false reactive results). 7 RTs brands (2 lots of each brand) were selected for the verification study. The verification panel of 230 samples of blood sera, which does not contain HIV serological markers, was prepared from the sample archive of the laboratory of epidemiology of parenteral viral hepatitis and HIV infection of the SI «The L. V. Hromashevskyi institute of epidemiology and infectious diseases of NAMS of Ukraine». The testing of the samples of the verification panel took place in a random order and in a blind way — in the first half of the working day (from morning to lunch), 5 serum samples were tested for 2 lots of all 7 RTs brands in exact accordance with the instructions for using the tests. For the visual interpretatation of the testing results with rapid tests, the intensity scale of the test line was used, according to which the results of the study were evaluated. The visual interpretation of the results, taking into account their subjective evaluation, was carried out by two interpreters independently of each other (without their knowledge of the other version of the evaluation of the results and the status of the sample itself from the verification panel). When determining the general status of each sample, the results obtained on both lots of a specific RT were taken into account. The match of the result was considered final; in case of discordant results, the HIV-reactive result was taken into account. Results. During the evaluation, 230 sera samples of the verification panel were examined using 7 RTs brands (two lots of each RT brand). Invalid test results (absence of a control strip in the presence or absence of a test strip) were not recorded. All 230 samples from the verification panel were negative when tested by both lots of five RTs brands, so their 100% specificity was confirmed. False-reactive results were obtained for the remaining two RTs brands when 7 negative samples were tested: 6 samples were tested by one RT brand and 1 sample was tested by another RT brand. At the same time, no false cross-reactive results were recorded when using these test brands. According to the obtained data, the specificity of the one RT brand was 97.5% with 95% CI (95.5%–99.5%), the other RT brand — 99.6% (95% CI (98.8%–100%)). Conclusions. According to the results of tests with samples of the verification panel, no false cross-reaction was recorded for all RT brands. Th,e one RT brand with the highest number of false-reactive results can be offered for selection only as a first-line test in testing algorithms, while the other six RT brands can be used as first- and second-/third-line tests. The obtained data confirm the effectiveness of the use of RT in HIV testing algorithms, which are currently used in Ukraine for different contingents of the population - both representatives of the population and key groups. Evaluation of the effectiveness of HIV testing algorithms in the country should be periodically repeated using diagnostic kits that have different antigenic components or are based on different principles of action. Key words: verification, HIV testing, rapid tests.

A rare case of isoniazid mono-resistant tuberculosis presenting as cardiac tamponade along with an anterior mediastinal mass in a 15-year-old immunocompetent patient

ABSTRACT A 15-year-old boy presented with a sudden onset of breathlessness for 7 days, gradual loss of weight of 17.6 lbs over the last month and progressive hoarseness of voice for 7 months. The contrast-enhanced computed tomography (CECT) scan revealed a heterogeneously enhancing lesion in the anterior mediastinum with multiple discrete lymph nodes in the cervical and mediastinal locations. The GeneXpert MTB/RIF assay performed on the CT-guided biopsy of the mass was negative, but the culture for Mycobacterium tuberculosis was positive at 7 weeks of incubation. There was a suboptimal radiological response after 6 months of treatment. First-line drug susceptibility testing (DST) performed by line probe assay (LPA) on the positive culture detected high-level resistance to isoniazid. The treatment was modified as per DST results to which the patient responded well.

Questionable Validity of Creatinine-Based eGFR in Elderly Patients but Cystatin C Is Helpful in First-Line Diagnostics.

The recommended chronic kidney disease (CKD) first-line diagnostic test is based on the creatinine-derived (estimated) glomerular filtration rate (eGFR). Cystatin C use may provide a better assessment. We compared creatinine- and cystatin C-derived eGFR determination as the first-line diagnostic test for 112 hospital patients aged > 60 years (median = 76 years). The patients were judged to not have CKD (no-CKD group) according to the first-line diagnostic recommendations (n = 61, eGFR (CKD Epidemiology Collaboration (CKD-EPI)) ≥ 60 mL/min/1.73 m2, total urine protein < 150 mg/g creatinine, urinary red/white blood cells not increased) or classified to be at risk for kidney insufficiency due to aortic valve dysfunction (at-risk group; n = 51). The accuracy of the eGFR values was evaluated retrospectively with the final case diagnoses. The eGFR (Caucasian, Asian, pediatric, and adult formula (CAPA)) was found to be linearly correlated to the eGFR (CKD-EPI) (R2 = 0.5, slope = 0.69, p < 0.0001). In 93/112 (>80%) cases, the eGFR (CAPA) yielded lower values (on average ≈-20%). In 55/112 (49%) cases, the cystatin C-derived CKD stage was lower. CKD reclassification from no-CKD to a kidney-insufficient state (i.e., CKD1/2 to CKD3a/b or 4) or reclassification to a more severe kidney insufficiency (i.e., CKD3a → 3b/4 or 3b → 4) was found in 41/112 (37%) cases. A worse CKD classification (no-CKD → kidney-insufficient) based on the eGFR (CAPA) was plausible in 30% of cases in light of the final case diagnoses. In elderly patients (>60 years), renal function appears to be systematically overestimated by the creatinine-based eGFR (CKD-EPI), indicating that, for this group, the cystatin C-based eGFR (CAPA) should be used as the first-line diagnostic test.

Performance of clinical metagenomics in France: a prospective observational study

Metagenomic next-generation sequencing (mNGS) allows untargeted identification of a broad range of pathogens, including rare or novel microorganisms. Despite the recognition of mNGS as a valuable diagnostic tool for infections, the most relevant indications for this innovative strategy remain poorly defined. We aimed to assess the determinants of positivity and clinical utility of mNGS. In this observational study, we prospectively performed short-read shotgun metagenomics analysis as a second-line test (in cases of negative first-line test or when the symptoms were not fully explained by initial positive results) or as a first-line test in life-threatening situations requiring urgent non-targeted pathogen identification at the Necker-Enfants Malades Hospital (Paris, France). All sample types, clinical indications, and patient populations were included. Samples were accompanied by a mandatory form completed by the senior clinician or pathologist, on which the clinical level of suspected infection (defined as high or low) was indicated. We assessed the variables (gender, age, immune status, initial suspicion of infection, indication, and sample type) associated with mNGS pathogen detection using odds ratios (ORs) from multivariate logistic regression. Additional investigations were carried out using specific PCR or culture techniques, to confirm positive mNGS results, or when infectious suspicion was particularly high despite a negative mNGS result. Between Oct 29, 2019, and Nov 7, 2022, we analysed 742 samples collected from 523 patients. The initial suspicion of infection was either high (n=470, 63%) or low (n=272, 37%). Causative or possibly causative pathogens were detected in 117 (25%) samples from patients with high initial suspicion of infection, versus nine (3%) samples analysed to rule out infection (OR 9·1, 95% CI 4·6-20·4; p<0·0001). We showed that mNGS had higher odds of detecting a causative or possibly causative pathogenic virus on CNS biopsies than CSF samples (4·1, 1·7-10·7; p=0·0025) and in samples from immunodeficient compared with immunocompetent individuals (2·4, 1·4-4·1; p=0·0013). Concordance with conventional confirmatory tests results was 103 (97%) of 106, when mNGS detected causative or possibly causative pathogens. Altogether, among 231 samples investigated by both mNGS and subsequent specific tests, discordant results were found in 69 (30%) samples, of which 58 (84%) were mNGS positive and specific tests negative, and 11 (16%) mNGS negative and specific tests positive. Major determinants of pathogen detection by mNGS are immune status and initial level of suspicion of infection. These findings will contribute, along with future studies, to refining the positioning of mNGS in diagnostic and treatment decision-making algorithms. Necker-Enfants Malades Hospital and Institut Pasteur. For the French translation of the abstract see Supplementary Materials section.

Safe Deferral of Coronary Computed Tomography Angiography for Patients With a Low Pretest Probability of Coronary Artery Disease in 2019 European Society of Cardiology Guidelines.

Coronary computed tomography angiography is increasingly used as the first-line test for suspected coronary artery disease. Its overuse in a low pretest probability (PTP) population may lead to low diagnostic yield without change in patient management. We evaluated the clinical consequences of the updated 2019 European Society of Cardiology (ESC) chronic coronary syndromes guidelines' PTP estimation and whether imaging could be safely deferred in patients with a low PTP. This retrospective cohort included all 1753 consecutive patients who underwent coronary computed tomography angiography for suspected coronary artery disease at Helsinki University Hospital between 2009 and 2017. PTP was calculated according to the 2013 and 2019 ESC guidelines. The overall mortality, cardiac deaths, myocardial infarctions, and hospitalizations for unstable angina were acquired from national registry data for 1 to 10 years of follow-up (median, 4 years). Updated 2019 ESC guidelines classified 72% of the patients as having low PTP, whose imaging could have been deferred. The revascularization rate (4.7%) and annual cardiac mortality (0.4%) were low in the 857 patients reclassified from the recommendation to test, according to the 2013 ESC guideline, to deferral of coronary computed tomography angiography, according to the new 2019 guideline. The updated 2019 ESC guideline PTP score aids clinicians in safely preventing the overuse of cardiac imaging in patients deemed at low PTP of coronary artery disease. Diagnostic yield, revascularization rate, and cardiac mortality are low in patients with low pretest risk.

Clinical application of bladder MRI and the Vesical Imaging-Reporting And Data System.

Diagnostic work-up and risk stratification in patients with bladder cancer before and after treatment must be refined to optimize management and improve outcomes. MRI has been suggested as a non-invasive technique for bladder cancer staging and assessment of response to systemic therapy. The Vesical Imaging-Reporting And Data System(VI-RADS) was developed to standardize bladder MRI image acquisition, interpretation and reporting and enables accurate prediction of muscle-wall invasion of bladder cancer. MRI is available in many centres but is not yet recommended as a first-line test for bladder cancer owing to a lack of high-quality evidence. Consensus-based evidence on the use of MRI-VI-RADS for bladder cancer care is needed to serve as a benchmark for formulating guidelines and research agendas until further evidence from randomized trials becomes available.

Assessment of Optical Genome Mapping for Front-Line Diagnostic Evaluation of Acute Leukemia: A Canadian Single-Center Evaluation of Added Yield in 69 Informative Cases

Optical genome mapping (OGM) is a novel method currently being evaluated internationally as a diagnostic assay for genomic profiling of malignancies. OGM uses imaging and reference alignment of fluorescently labeled ultra-long DNA molecules to detect structural and copy number variants genome-wide at significantly higher resolution than is attainable with current standard-of-care (SOC) cytogenetic analysis. OGM is nearing deployment as a first-line diagnostic test for patients with acute leukemia at Vancouver General Hospital, the largest tertiary care hospital in British Columbia. Here, we describe our experience in the evaluation of OGM as a diagnostic assay relative to SOC karyotyping and fluorescence in situ hybridization (FISH) in newly diagnosed and relapsed acute leukemia. A selected cohort of 69 bone marrow specimens produced adequate quality OGM data for analysis: 52 acute myeloid leukemia (AML), 12 B-lymphoblastic leukemia (B-ALL), 4 mixed phenotype acute leukemia (MPAL; 2 B/myeloid, 1 B/T, and 1 T/myeloid), and 1 blast phase (T) myeloid/lymphoid neoplasm with tyrosine kinase gene fusion (M/L-N-Eo). Variants were filtered using population thresholds and with pan-cancer (669 genes) and disease targeted (myeloid: 185; B-lymphoid: 130) gene lists to facilitate detection of variants of interest. OGM results were compared to SOC to identify additional diagnostic yield and changes to risk stratification or diagnosis resulting from OGM findings. In these 69 acute leukemias, filtering to remove common polymorphisms using a manufacturer-provided control dataset yielded a median of 53 variants (interquartile range (IQR) 39-86); using a pan-cancer list yielded a median of 6 (IQR 2-16); and disease-specific lists a median of 4 (IQR 1-10.5). In 11% of cases, diagnosis or risk stratification was altered by OGM (13% of AML; 8% of B-ALL), including a complex karyotype being resolved as normal, downgrading cytogenetic risk; identification of several cryptic KMT2A rearrangements, changing diagnostic classification; and identification of cryptic MECOM::RUNX1 (class-modifying), NUP98::NSD1 (risk-upgrading), and NUP214::ABL1 (potentially targetable and class- and risk-modifying) fusions. In 26% of cases, driver rearrangements were either newly identified by OGM or novel partner genes were resolved (23% of AML; 33% of B-ALL; 40% of others), including recurrent rearrangements of the IGH locus with MIR125B1/ BLID in B-ALL; KMT2A partial tandem duplication; novel MECOM::PAN3, JAKMIP2::PDGFRB, and NUP214::FRMD4B fusions; and a cryptic deletion juxtaposing FLT3 and PAN3 known to result in FLT3 overexpression. Additional findings, including copy number changes and clarifications of complex events, were seen in 54% of cases (44% of AML; 83% of B-ALL; 80% of others), mostly accounted for by small deletions or disruptive rearrangements in key disease genes,likely resulting in loss of function, including but not limited to RUNX1, GATA2 and ETV6 in AML; CDKN2A, PAX5, and RB1 in B-ALL; and TP53 in MPAL. Several cases harboured small MLLT4 copy number abnormalities that have been reported to be linked to cancer predisposition, and one potentially germline FANCA deletion was observed. Details of comparison between SOC methods (karyotyping and pertinent FISH where applicable) are detailed in the Figure and Table (bold text: newly resolved or clarified potential driver or resolution of normal karyotype; underline: change in diagnosis or risk stratification resulting from OGM findings). Notably, OGM failed to identify one hypodiploid B-ALL clone masked by endoreduplication, and further evaluation of this technology's performance in this context and in prospective practice is required. This study demonstrates the significant potential added diagnostic yield of OGM relative to SOC as a clinical diagnostic and research assay. High-resolution genome-wide evaluation not reliant on cell culture substantially expands the scope of detectable variants, for many of which determining the biologic and clinical implications will require larger scale prospective study. Importantly, application of OGM in the clinical setting is likely to result in clinically meaningful disease reclassification and restratification of risk, and in some cases may permit patients access to previously inaccessible targeted therapies (as in the instance of cytogenetically cryptic tyrosine kinase gene fusions).

Women's preferences for NIPT as a first-line test in England and France: Challenges for genetic counseling practices.

Non-invasive prenatal testing (NIPT) is provided in the private and public sectors worldwide as a first- or second-tier test. In England and France, NIPT is fully funded and offered as a contingent strategy with different probability cut-offs (1:150 and 1:1000). These different approaches to define the target population for NIPT have implications for how women experience their antenatal care. The paper explores and compares the perceptions and difficulties of women in England and France who took NIPT as a second-tier screening test. It is based on a semi-structured qualitative interview study with 17 women in England and France conducted between September 2021 and May 2022. The interviews were cross-analyzed using thematic analysis. Our findings show that most women express a preference for the offer of NIPT as a first-line screening test. Some issues with the contingent model, related to the access to information and termination of pregnancy (TOP), the disparities of NIPT uptake, and risks of generating anxiety with combined first-trimester screening (cFTS), could be addressed by a universal strategy for T21, T13, and T18. Nevertheless, this strategy could present some challenges for genetic counseling due to: women's understanding and expectations of NIPT; adequate information and counseling about the scope and limits of NIPT; concerns about the routinization of NIPT in the first-line offer; limitations and uncertainties associated with the provision of expanded NIPT in France; the remaining importance of other screening tests; and associated costs.

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases

BackgroundWhole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25–30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome.MethodsWe undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants.ResultsOur diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving.ConclusionsGenome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.