Next-Generation Multitarget Stool DNA Test for Colorectal Cancer Screening.

BACKGROUND: A next-generation multitarget stool DNA test, including assessments of DNA molecular markers and hemoglobin level, was developed to improve the performance of colorectal cancer screening, primarily with regard to specificity. METHODS: In a prospective study, we evaluated a next-generation multitarget stool DNA test in asymptomatic adults 40 years of age or older who were undergoing screening colonoscopy. The primary outcomes were sensitivity of the test for colorectal cancer and specificity for advanced neoplasia (colorectal cancer or advanced precancerous lesions). Advanced precancerous lesions included one or more adenomas or sessile serrated lesions measuring at least 1 cm in the longest dimension, lesions with villous histologic features, and high-grade dysplasia. Secondary objectives included the quantification of sensitivity for advanced precancerous lesions and specificity for nonneoplastic findings or negative colonoscopy and comparison of sensitivities for colorectal cancer and advanced precancerous lesions between the multitarget stool DNA test and a commercially available fecal immunochemical test (FIT). RESULTS: Of 20,176 participants, 98 had colorectal cancer, 2144 had advanced precancerous lesions, 6973 had nonadvanced adenomas, and 10,961 had nonneoplastic findings or negative colonoscopy. With the next-generation test, sensitivity for colorectal cancer was 93.9% (95% confidence interval [CI], 87.1 to 97.7), and specificity for advanced neoplasia was 90.6% (95% CI, 90.1 to 91.0). Sensitivity for advanced precancerous lesions was 43.4% (95% CI, 41.3 to 45.6), and specificity for nonneoplastic findings or negative colonoscopy was 92.7% (95% CI, 92.2 to 93.1). With the FIT, sensitivity was 67.3% (95% CI, 57.1 to 76.5) for colorectal cancer and 23.3% (95% CI, 21.5 to 25.2) for advanced precancerous lesions; specificity was 94.8% (95% CI, 94.4 to 95.1) for advanced neoplasia and 95.7% (95% CI, 95.3 to 96.1) for nonneoplastic findings or negative colonoscopy. As compared with FIT, the next-generation test had superior sensitivity for colorectal cancer (P<0.001) and for advanced precancerous lesions (P<0.001) but had lower specificity for advanced neoplasia (P<0.001). No adverse events occurred. CONCLUSIONS: The next-generation multitarget stool DNA test showed higher sensitivity for colorectal cancer and advanced precancerous lesions than FIT but also showed lower specificity. (Funded by Exact Sciences; BLUE-C ClinicalTrials.gov number, NCT04144738.).

Evaluation of a new Electrochemiluminescence Immunoassay System for Measuring High-Sensitivity Cardiac Troponin T.

BACKGROUND: The aim of this study was to verify the analytical performance of the UD90DT electrochemiluminescence immunoassay system (the UD90DT system) for measuring high-sensitivity cardiac troponin T (hs-cTnT). METHODS: According to the Clinical and Laboratory Standards Institute guidelines, the imprecision, linearity, reference interval, limit of blank (LoB), limit of detection (LoD), and functional sensitivity (FS) of hs-cTnT using the UD90DT system were verified. The trueness was validated using the Proficiency Testing (PT) materials. RESULTS: The within-run and between-run coefficients of variations (CVs) of two hs-cTnT levels were 7.2% and 1.5%, and 7.1% and 2.6%, respectively. The biases of the PT samples (n = 6) all fell within the allowable total error. The linearity satisfied the requirements, with a slope of 0.9963 and an R12 value of 0.9998. The hs-cTnT levels of the healthy volunteers (n = 20) ranged from 3.0 ng/L to 7.7 ng/L. All blank calibrator measurements (n = 20) fell within the LoD claim, and none of the samples (n = 25) had a LoB value ≤ 3.0 ng/L. The FS was 5.3 ng/L. Furthermore, a good correlation between the UD90DT system and the Cobas e 601 module was observed for hs-cTnT. CONCLUSIONS: The analytical performance of hs-cTnT using the UD90DT system is acceptable and satisfies clinical needs.

Quantitative and Standardized Pseudovirus Neutralization Assay for COVID-19.

COVID-19 is a global pandemic caused by the highly infectious SARS-CoV-2 virus. Efforts to combat SARS-CoV-2 infection include mass vaccination and development of monoclonal and convalescent plasma therapeutics that require precise measurements of correlative, functional neutralizing antibodies that prevent virus infection. Developing rapid, safe, easy-to-use, and high-quality neutralization assays are essential for the success of the massive effort. Here, we developed a vesicular stomatitis virus-based neutralization assay that was capable of quantifying varying degrees of neutralization in patient serum samples. This assay has two detection readouts, flow cytometry and live cell imaging. The two readout methods produced consistent values of all 50% neutralization titers, further enhancing measurement confidence on the assay. Moreover, the use of available reference standards such as the World Health Organization International Standard (NIBSC code 20/136) enables quantification and standardization of the pseudovirus neutralization assay with neutralizing antibody titers measured in International Units/mL. Quantitative and standardized neutralization assays are critical for reliable efficacy evaluation and comparison of numerous vaccines and therapeutics.

CyTOF Intracellular Cytokine Assays for Antigen-Specific T Cells.

T cells specific for a single antigen tend to be rare, even after expansion of memory cells. They are commonly detected by in vitro stimulation with peptides or protein, followed by staining for intracellular cytokines. In this protocol, CyTOF® mass cytometry is used to collect single-cell data on a large number of cytokines/chemokines, as well as cell-surface proteins that characterize T cells and other immune cells. A method for magnetic bead enrichment of antigen-stimulated T cells, based on their expression of CD154 and CD69, is also included. Coupling magnetic enrichment with highly multiparameter mass cytometry, this method enables the ability to dissect the frequency, phenotype, and function of antigen-specific T cells in greater detail than previously possible. Rare cell subsets can be examined, while minimizing run times on the CyTOF.

Total IgE as a biomarker of omalizumab response in chronic spontaneous urticaria: A meta-analysis.

Background: Omalizumab is approved for the treatment of chronic spontaneous urticaria (CSU) that is refractory to antihistamines. Total immunoglobulin E (IgE) levels have emerged as a possible biomarker to predict response to omalizumab. However, the existing literature is heterogenous, with conflicting conclusions with regard to the role of total IgE levels. Objective: We sought to clarify the role of evaluating total IgE levels in patients with CSU by performing a meta-analysis on the existing literature to determine if meaningful changes exist between responders and nonresponders to omalizumab. Methods: A total of 68 unique citations were returned and screened by two independent reviewers. Editorials, reviews, and case reports were excluded, and a total of 33 original articles were identified and underwent secondary evaluation. Studies that present mean ± standard deviation total IgE levels and/or 95% confidence intervals (CI) were included, whereas studies with < 25 subjects were excluded. Three studies ultimately met these criteria. Results: We found a mean difference in total IgE levels between those who responded to omalizumab versus those without a response of 49.76 (95% CI, 7.13-92.38; p = 0.02), which demonstrated higher mean IgE values in responders compared with nonresponders. Conclusion: This study presents additional evidence that supports evaluation of total IgE levels as it pertains to response to omalizumab therapy in CSU. When considering the current evidence, it seems reasonable to consider the baseline total IgE level as a biomarker to predict the treatment response to omalizumab. Based on the existing literature, we cannot conclude at what threshold nonresponse is more likely to occur.

Diagnostic accuracy of the IFN-γ release assay using RD1 immunodominant T-cell antigens for diagnosis of extrapulmonary tuberculosis.

The diagnosis of extrapulmonary tuberculosis (EPTB) poses a significant challenge, with controversies surrounding the accuracy of IFN-γ release assays (IGRAs). This study aimed to assess the diagnostic accuracy of RD1 immunodominant T-cell antigens, including ESAT-6, CFP-10, PE35, and PPE68 proteins, for immunodiagnosis of EPTB. Twenty-nine patients with EPTB were enrolled, and recombinant PE35, PPE68, ESAT-6, and CFP-10 proteins were evaluated in a 3-day Whole Blood Assay. IFN-γ levels were measured using a Human IFN-γ ELISA kit, and the QuantiFERON-TB Gold Plus (QFT-Plus) test was performed. Predominantly, the patients were of Afghan (62%, n = 18) and Iranian (38%, n = 11) nationalities. Eighteen individuals tested positive for QFT-Plus, accounting for 62% of the cases. The positivity rate for IGRA, using each distinct recombinant protein (ESAT-6, PPE68, PE35, and CFP-10), was 72% (n = 21) for every protein tested. Specifically, among Afghan patients, the positivity rates for QFT-Plus and IGRA using ESAT-6, PPE68, PE35, and CFP-10 were 66.7%, 83.3%, 83.3%, 77.8%, and 88.9%, respectively. In contrast, among Iranian patients, the positivity rates for the same antigens were 54.5%, 54.5%, 54.5%, 63.6%, and 45.5%, respectively. In conclusion, our study highlights the potential of IGRA testing utilizing various proteins as a valuable diagnostic tool for EPTB. Further research is needed to elucidate the underlying factors contributing to these disparities and to optimize diagnostic strategies for EPTB in diverse populations.

Comparison of immunological and molecular methods for laboratory diagnosis of ocular toxoplasmosis in blood, serum and tears in Brazil.

Ocular toxoplasmosis (OT) is caused by protozoan T. gondii. Ophthalmological examination is considered the gold standard for OT diagnosis, and laboratory tests are used for diagnostic confirmation. However, these tests can present different results, which change depending on their basis, on sample type and on patients' clinical alteration. Thus, the aim of the present study is to assess immunodiagnostic and molecular techniques applied in blood, serum and tear fluid to diagnose T. gondii infection in patients seen at an Ophthalmology Clinic. In total, 160 patients were included in the study, 40 of them had OT with active lesions (G1); 40 had OT with healed lesions (G2), 40 had non-toxoplasmic uveitis (G3) and 40 had no ocular alterations (G4). Serum samples were subjected to Immunoenzymatic Assay (ELISA) and to Indirect Immunofluorescence Reaction (IFAT) to search for anti-T. gondii IgM and IgG. Tear fluid samples were analyzed through ELISA for IgA research. All blood and tear fluid samples were subjected to conventional polymerase chain reaction (cPCR) and in a Nested PCR model for T. gondii DNA amplification with targets B1, GRA7 and REP 529. IgG and IgM anti-T. gondii was detected in serum samples from 106 and 15 patients, respectively, when combining ELISA and IFAT results. Anti-T.gondii IgA antibodies were detected in 9.2% of the tear material. Nested PCR with GRA7 target showed higher positivity in blood samples (24.4%); Nested PCR with B1 target showed a higher frequency of positivity in tears (15%). Biological samples of patients with active lesions showed the highest positivity frequencies in all immunodiagnostic assays, as well as in most PCR models. The present results highlighted the need of associating techniques with different fundamentals to confirm OT diagnosis. Furthermore, further tear fluid analyses should be performed to validate this biological material as lesser invasive alternative for the more accurate OT diagnosis.

Rapid antigen testing for SARS-CoV-2 by lateral flow assay: A field evaluation of self- and professional testing at UK community testing sites.

BACKGROUND: The advent of lateral flow devices (LFDs) for SARS-CoV-2 detection enabled widespread use of rapid self-tests during the pandemic. While self-testing using LFDs is now common, whether self-testing provides comparable performance to professional testing was a key question that remained important for pandemic planning. METHODS: Three prospective multi-centre studies were conducted to compare the performance of self- and professional testing using LFDs. Participants tested themselves or were tested by trained (professional) testers at community testing sites in the UK. Corresponding qRT-PCR test results served as reference standard. The performance of Innova, Orient Gene and SureScreen LFDs by users (self) and professional testers was assessed in terms of sensitivity, specificity, and kit failure (void) rates. Impact of age, sex and symptom status was analysed using logistic regression modelling. RESULTS: 16,617 participants provided paired tests, of which 15,418 were included in the analysis. Self-testing with Innova, Orient Gene or SureScreen LFDs achieved sensitivities of 50 %, 53 % or 72 %, respectively, compared to qRT-PCR. Self and professional LFD testing showed no statistically different sensitivity with respect to corresponding qRT-PCR testing. Specificity was consistently equal to or higher than 99 %. Sex and age had no or only marginal impact on LFD performance while sensitivity was significantly higher for symptomatic individuals. Sensitivity of LFDs increased strongly to up to 90 % with higher levels of viral RNA measured by qRT-PCR. CONCLUSIONS: Our results support SARS-CoV-2 self-testing with LFDs, especially for the detection of individuals whose qRT-PCR tests showed high viral concentrations.

Infrequent patterns in cerebrospinal fluid isofocusing test: Clinical significance and contribution of IgG index and Reiber diagram to their interpretation.

Cerebrospinal fluid (CSF) isoelectrofocusing (IEF) is considered as the gold standard for detecting an intrathecal synthesis of IgG, which is a hallmark of multiple sclerosis (MS). This corresponds to the presence of CSF-restricted IgG oligoclonal bands (OCB) (typically type 2 pattern). Moreover, this technique can also detect a systemic immune reaction with passive transfer of IgG (type 3 and 4 patterns) for which the clinical relevance is less understood. The aim of our study was to determine the frequency and disease associations of IEF type 3 and 4 patterns and to investigate the potential usefulness of including quantitative data (IgG index and Reiber Diagram) in interpreting such IEF profiles. Among 544 patients who underwent CSF IEF (Hydragel CSF isofocusing kit, Sebia®, France) in our Laboratory during a six-year-period, those who presented type 3 or 4 patterns were selected. Clinical data and results of other immunological tests were analyzed for 27 patients followed in the Neurological Department. Frequencies of type 3 and type 4 patterns were relatively low (2.3 % and 3.4 % respectively). Among patients with type 3 pattern included in our study (n = 10), 5 were diagnosed with MS. For the 5 other patients, the diagnosis was a clinically isolated syndrome (CIS) (2 cases), a probable auto-immune encephalitis (2 cases) and a possible genetic neurodegenerative disease (1 case). MS patients had an IgG index >0.7 and fell into area 4 of Reiber diagram while non-MS patients had an IgG index <0.7 and fell into area 1, except the last case. Regarding type 4 pattern (n = 17), the diagnoses were as follows: MS (3), CIS (4), Neuromyelitis optica spectrum disorders with positive anti-AQP4 antibodies (3) and anti-NMDAR autoimmune encephalitis (1). The remaining cases had central nervous system impairment related to vascular, metabolic or tumoral etiologies (3) or peripheral nervous system impairment (3). In this group (type 4 pattern), IgG index was <0.7 in 15/17 cases. Interpretation using Reiber diagram showed an abnormal blood-brain barrier for 8/17 patients. Type 3 and 4 IEF patterns are infrequently observed in routine neurology practice. It is important for the diagnostic laboratory professional as well as for the neurologist to understand their clinical relevance. Our findings highlight the contribution of quantitative evaluation of CSF (IgG index, Reiber diagram) for the interpretation of such situations. Despite the small size of our study population, our results emphasize the importance of reporting the exact type of IEF pattern and not only the positivity or not of OCB.

Clinical performance of AMA-M2, anti-gp210 and anti-sp100 antibody levels in primary biliary cholangitis: When detected by multiplex bead-based flow fluorescent immunoassay.

BACKGROUND AND AIM: Primary biliary cholangitis (PBC) is a chronic autoimmune cholangiopathy, characterized by the presence of some autoantibodies in the serum. This study aimed to evaluate the clinical significance of AMA-M2, anti-gp210 and anti-sp100 antibody levels detected by multiplex bead-based flow fluorescent immunoassay (MBFFI) in PBC. METHODS: This study cohort included 238 PBC patients, 81 autoimmune hepatitis (AIH) patients, 62 systemic lupus erythematosus (SLE) patients, and 118 healthy controls. Serum AMA-M2, anti-gp210 and anti-sp100 antibody were detected by MBFFI and immunoblotting assay (IBT). The relationship between three antibody levels and cirrhosis, liver function, cholestasis markers and therapeutic effect to ursodesoxycholic acid (UDCA) was evaluated in PBC. RESULTS: MBFFI were presented good coincidence rate (87.39%-95.38%) with IBT. The level of AMA-M2, anti-gp210 and anti-sp100 antibodies in PBC patients were higher than other disease group and healthy controls (p ＜ .01). When compared with the healthy controls group, the AUC of AMA-M2, anti-gp210 and anti-sp100 antibodies were 0.9245, 0.7619, and 0.6789, respectively. In addition, gp210 antibody levels have diagnostic value in patients with liver cirrhosis (AUC: 0.7567). We found that when combine detect these three antibodies, the sensitivity was higher than individually detection. High level of serum anti-gp210 antibody could be related to worse liver function and more severe cholestasis in PBC patients. Moreover, serum antibody levels may decrease or remained flat in patients who responded well to UDCA. CONCLUSION: The detection of AMA-M2, anti-gp210 and anti-sp100 antibody levels by MBFFI showed good performance in the diagnosis of PBC. Serum anti-gp210 antibody level is related to cirrhosis, poor liver function and severe cholestasis in PBC.

Developmental Toxicity Using the Rat Whole Embryo Culture.

In vivo and in vitro experiments have been used to investigate the effect of drugs, chemical agents, growth factors, vitamins, etc., on embryonic development. Alternative tests have been developed to determine whether drugs or other compounds have toxic or teratogenic effects on a particular organ or tissue. The rat whole embryo culture method is useful for studying the mechanism of normal embryo development. The explanted rat conceptuses grow in the culture environment at the same rate as in utero development. Furthermore, the considerable advantage of explanting rat embryos is that it allows direct observation of embryogenesis. The rat whole embryo culture is run by explanting rat embryos on gestation day 9.5 for rats. The conceptuses are then cultured on a rotating platform in a mixture of culture medium with appropriate gassing for 48 hours. The maternal serum is used as the culture medium, and chemicals or other agents to be evaluated separately are added to this medium. At the end of the culture period, growth and development of the embryos are evaluated morphologically.

Double-Amplified Electrochemiluminescence Immunoassay Sensor for Highly Sensitive Detection of CA19-9 Using a Ternary Semiconductor CdSSe.

In this paper, an electrochemiluminescence (ECL) immunosensor for ultrasensitive detection of CA19-9 was constructed using ternary compound CdSSe nanoparticles as ECL emitter. The immunosensor employs Cu2S and gold-doped diindium trioxide (Au-In2O3) nanocubes as coreaction accelerators to achieve a double-amplification strategy. In general, a hexagonal maple leaf-shaped Cu2S with a large surface area was selected as the template, and the in situ growth of CdSSe on its surface was achieved using a hydrothermal method. The presence of Cu2S not only inhibited the aggregation of CdSSe nanoparticles to reduce their surface energy but also acted as an ECL cathode coreaction promoter, facilitating the generation of SO4•-. Consequently, the ECL intensity of CdSSe was significantly enhanced, and the reduction potential was significantly lower. In addition, the template method was employed to synthesize Au-In2O3 nanocubes, which offers the advantage of directly connecting materials with antibodies, resulting in a more stable construction of the immunosensor. Furthermore, In2O3 serves as a coreaction promoter, enabling the amplification strategy for ECL intensity of CdSSe, thus contributing to the enhanced sensitivity and performance of the immunosensor. The constructed immunosensor exhibited a wide linear range (100 µU mL-1 to 100 U mL-1) and a low detection limit of 80 µU mL-1, demonstrating its high potential and practical value for sensitive detection of CA19-9.

Single-Molecule Protein Analysis by Centrifugal Droplet Immuno-PCR with Magnetic Nanoparticles.

Detecting proteins in ultralow concentrations in complex media is important for many applications but often relies on complicated techniques. Herein, a single-molecule protein analyzer with the potential for high-throughput applications is reported. Gold-coated magnetic nanoparticles with DNA-labeled antibodies were used for target recognition and separation. The immunocomplex was loaded into microdroplets generated with centrifugation. Immuno-PCR amplification of the DNA enabled the quantification of proteins at the level of single molecules. As an example, ultrasensitive detection of α-synuclein, a biomarker for neurodegenerative diseases, is achieved. The limit of detection was determined to be ∼50 aM in buffer and ∼170 aM in serum. The method exhibited high specificity and could be used to analyze post-translational modifications such as protein phosphorylation. This study will inspire wider studies on single-molecule protein detection, especially in disease diagnostics, biomarker discovery, and drug development.

Silver Nanocluster-Tagged Electrochemiluminescence Immunoassay with a Sole and Narrow Triggering Potential Window.

The commercialized electrochemiluminescence (ECL) immunoassay is carried out by holding luminophore Ru(bpy)32+ at a given potential. Designing an electrochemiluminophore with a narrow triggering potential window is strongly anticipated to decrease the electrochemical cross-talk and improve the flux of the commercialized ECL immunoassay in a potential-resolved way. Herein, L-penicillamine-capped silver nanoclusters (LPA-AgNCs) are facilely synthesized and utilized as tags to perform the ECL immunoassay with a sole and narrow triggering potential window of 0.24 V by employing hydrazine (N2H4) as a coreactant. The maximum ECL emission of the LPA-AgNCs/N2H4 system is located ca. +1.27 V. Upon immobilizing LPA-AgNCs onto the electrode surface via forming a sandwich immunocomplex, the ECL of LPA-AgNCs/N2H4 can be utilized to sensitively and selectively determine human carcinoembryonic antigen from 0.5 to 1000 pg/mL with a low limit of detection of 0.1 pg/mL (S/N = 3). This work might open a way to screen electrochemiluminophores for the multiple ECL immunoassay in a potential-resolved way.

Clinical evaluation of a novel plasma pTau217 electrochemiluminescence immunoassay in Alzheimer's disease.

A growing literature suggests that plasma levels of tau phosphorylated at amino acid 217 (pTau217) performs similarly to cerebrospinal fluid (CSF) biomarkers and PET imaging to detect amyloid pathology and to provide diagnostic and prognostic information in Alzheimer's disease (AD), but a significant limiting factor thus far has been a lack of widely available immunoassays. We evaluated a novel pTau217 S-PLEX® assay developed by Meso Scale Discovery (MSD; Rockville, MD) in plasma from 131 individuals with AD confirmed by CSF biomarkers and controls. Technical performance of the assay was excellent with an LLOQ of 1.84 pg/mL and intra/interplate CVs of 5.5% (0.3-15.0%) and 5.7% (range 0.3-13.4%), respectively. The pTau217 plasma assay differentiated AD and controls with an AUC of 0.98 (95% CI 0.96-1.0) and pTau217 levels were 3.9-fold higher in individuals with AD. This performance was significantly better than what was observed for plasma pTau181, performed in parallel, and comparable to published data on existing pTau217 assays. While further clinical validation and head-to-head comparisons are needed to fully establish the role for the novel pTau217 S-PLEX assay, these data demonstrate the utility of the assay to detect AD pathology.

Improving diagnostic performance of coronavirus disease 2019 rapid antigen testing through computer-based feedback training using open-source experimental psychology software.

INTRODUCTION: Rapid antigen testing (RAT) results are visually read as whether colored line is present or absent. The subjective interpretation potentially misses detecting weak lines due to lower analyte concentration in samples tested, requiring training. Although routine test experience has improved the result readout skills, it consumes time and resources. Therefore, we created a computer-based feedback training method using open-source experimental psychology software, wherein participants accumulate RAT result readout experience by repeatedly responding positive/negative to randomly presented pictures showing RAT results; then, they receive feedback on their answers as correct or incorrect and are asked to stare at the pictures again with the knowledge of correct answer. This study aimed to examine the training effects in improving the skills, using coronavirus disease 2019 (COVID-19) RAT. METHODS: Twenty-two medical technologists were randomly divided into two groups: the feedback-training and test-experience groups. Using several pictures showing positive and negative results of COVID-19 RAT, after examination of their initial result readout skills, feedback-training group received the feedback training, whereas test-experience group performed an equal number of tests without feedback to accumulate test experience, and their skills were examined again. The ratio of "positive" answers to the pictures showing positive results (i.e., hit rate) was statistically analyzed. RESULTS: The feedback-training group showed a significantly higher hit rate after their training, whereas the test-experience group did not. The feedback training effects were manifested in weak line detection. CONCLUSIONS: This computer-based feedback training method can be an effective tool for improving RAT result readout skills.